Compositions and methods for enhanced expression of recombinant polypeptides from a single vector using a peptide cleavage site

ABSTRACT

Vector constructs for expression of two or more functional proteins or polypeptides under operative control of a single promoter and methods of making and using the same are described. The vectors comprise a self-processing cleavage site between each respective protein or polypeptide coding sequence. The vector constructs include the coding sequence for a self-processing cleavage site and may further include an additional proteolytic cleavage sequence which provides a means to remove the self processing peptide sequence from expressed protein(s) or polypeptide(s). The vector constructs find utility in methods for enhanced production of biologically active proteins and polypeptides in vitro and in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. patent applicationSer. No. 10/452,253, filed Jun. 3, 2003 and U.S. Provisional PatentApplication No. 60/540,553, filed Feb. 2, 2004. The priorityapplications are hereby incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The invention relates to novel vector or plasmid constructs designed toexpress recombinant polypeptides or fragments thereof by using aself-processing peptide. The constructs may be used for in vitro, exvivo or in vivo delivery of heterologous protein or polypeptide codingsequences to a cell, or in the production of recombinant polypeptides byvector-transduced or plasmid transfected cells.

BACKGROUND OF THE INVENTION

Recombinant proteins as therapeutic modalities have found increasing usein recent years. Numerous recombinant protein-based therapies are invarious stages of clinical development. One limitation to widespreadclinical application of recombinant protein technology is the difficultyin production of proteins that include two or more coding sequences ordomains such that the domains are expressed in the proper ratio withappropriate post-translational processing resulting in production of afunctional heterodimeric molecule. A further limitation is the high costassociated with adequate levels of expression for clinical applications.

Chinese Hamster Ovarian (CHO) cells are the most commonly used mammaliancell lines for commercial scale production of glycosylated humanproteins. Previous attempts to express a full length recombinant proteinwith two or more domains or chains (and thus two or more codingsequences or open reading frames (ORFs)) via recombinant DNA technologyusing a single vector have met with limited success, typically resultingin unequal levels of expression of the two or more domains or chains ofthe protein or polypeptide and more particularly, a lower level ofexpression for the second coding sequence. In order to express a fullybiological functional protein or polypeptide which has two or moredomains or chains from a single vector, equimolar expression of the twoor more domains or chains is typically required. Additionally,conventional vectors relying on dual promoter regulation of geneexpression are invariably affected by promoter interaction (i.e.,promoter interference) which may compromise equimolar or substantiallyequimolar expression of the genes. Other factors that limit the abilityto express two or more coding sequences from a single vector include thepackaging limitation of the vector itself. For example, in consideringthe appropriate vector/coding sequence combination, factors to beconsidered include: the capacity of the vector (e.g., approx. 4,500 bpfor AAV); the duration of expression of the recombinant molecule byvector-transduced cells (e.g., short term expression for adenoviralvectors); the cell types infected by the vector if a viral vector isused; and the desired expression level of the target gene product(s).The requirement for controlled expression of two or more gene productstogether with the packaging limitations of viral vectors such asadenovirus and AAV restrict the choices with respect to vectorconstruction and systems for expression of a protein or polypeptidewhich has two or more domains or chains.

The linking of proteins in the form of polyproteins in a single openreading frame is a strategy adopted in the replication of many virusesincluding picornaviridae. Upon translation, virus-encoded proteinasesmediate rapid intramolecular (cis) cleavage of a polyprotein to yielddiscrete mature protein products. Foot and Mouth Disease viruses (FMDV)are a group within the picornaviridae which express a single, long openreading frame encoding a polyprotein of approximately 225 kD. The fulllength translation product undergoes rapid intramolecular (cis) cleavageat the C-terminus of a self-processing cleavage site, for example, a 2Asite or region, located between the capsid protein precursor (P1-2A) andreplicative domains of the polyprotein 2BC and P3, with the cleavagemediated by proteinase-like activity of the 2A region itself (Ryan etal., J. Gen. Virol. 72:2727-2732, 1991); Vakharia et al., J. Virol.61:3199-3207, 1987). Similar domains have also been characterized fromaphthoviridea and cardioviridae of the picornavirus family (Donnelly etal., J. Gen. Virol. 78:13-21, 1997).

In order to express proteins or polypeptides which have two or moredomains or chains from a single vector, two or more promoters or aninternal ribosome entry site (IRES) sequence are generally used to driveexpression of individual genes. The use of two promoters within a singlevector can result in low protein expression, e.g., due to promoterinterference. When two genes are linked with an IRES sequence, theexpression level of the second gene is often significantly lower thanthe first gene (Furler et al., Gene Therapy 8:864-873, 2001).

There remains a need for improved gene expression systems for productionof recombinant proteins and polypeptides, in particular proteins andpolypeptides that have two or more domains or chains, such thatsufficient expression of a biologically active recombinant protein orpolypeptide is achieved at commercially reasonable cost.

The present invention addresses this need by demonstrating thefeasibility and use of a single vector or plasmid construct comprising asequence that encodes a self-processing peptide which results in theexpression of functional recombinant proteins and polypeptides.

SUMMARY OF THE INVENTION

The present invention provides a means for recombinant protein orpolypeptide expression using a self-processing peptide which facilitatesefficient expression of two or more polypeptides from a single openreading frame by providing a “cleavage” site to generate individualpolypeptides.

The present invention provides a system for expression of a protein orpolypeptide based on substantially equal expression of the codingsequence for two or more proteins or polypeptides or domains or chainsthereof under transcriptional control of the same promoter from a singlevector, wherein translation is mediated by a self-processing cleavagesite, e.g., a 2A or 2A-like sequence.

In one preferred aspect, the invention provides a vector or constructfor expression of two or more recombinant proteins or polypeptides or arecombinant protein or polypeptide which has two or more domains orchains (and thus two or more coding sequences or open reading frames(ORFs)). In an exemplary construct wherein 2 coding sequences areexpressed, the vector includes in the 5′ to 3′ direction: a promoteroperably linked to the coding sequence for a first protein orpolypeptide ORF, a sequence encoding a self-processing cleavage site andthe coding sequence for a second protein or polypeptide ORF, wherein thesequence encoding the self-processing cleavage site is inserted betweenthe coding sequence for the first protein or polypeptide and the codingsequence for the second protein or polypeptide.

The vector may be any recombinant vector capable of expression of aprotein or polypeptide of interest or a fragment thereof, for example,an adeno-associated virus (AAV) vector, a lentivirus vector, aretrovirus vector, a replication competent adenovirus vector, areplication deficient adenovirus vector (e.g., a gutless adenovirusvector), a herpes virus vector, a baculovirus vector or a nonviralplasmid.

Preferred self-processing cleavage sequences include a 2A sequence,e.g., a 2A sequence derived from Foot and Mouth Disease Virus (FMDV).

In a further preferred aspect, the vector comprises a sequence whichencodes an additional proteolytic cleavage site located between thecoding sequence for a first chain of a protein or polypeptide and thecoding sequence for a second chain of a protein or polypeptide, e.g., afurin cleavage site with the consensus sequence RXK(R)R (presented asSEQ ID NO:10).

A vector for recombinant protein or polypeptide expression using aself-processing peptide may include any of a number of promoters,wherein the promoter is constitutive, regulatable or inducible, celltype specific, tissue-specific, or species specific.

The vector may further comprise a signal sequence for the codingsequence of a domain of the protein or polypeptide.

In one preferred aspect of the invention, two or more protein orpolypeptide coding sequences are expressed in a substantially equimolarratio.

The invention further provides host cells transduced with a vector orplasmid that comprises that comprises a sequence encoding aself-processing cleavage site alone or in combination with a sequenceencoding an additional proteolytic cleavage site and use of such cellsin generating recombinant proteins or polypeptides.

In a related aspect, the invention provides a recombinant protein orpolypeptide produced by such a cell and methods for producing the same.

Other and further objects, features and advantages are apparent from thefollowing description of the embodiments for the invention given thepurpose of disclosure when taken in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D depict exemplary plasmids for the expression of solubleplatelet factor 4 (sPF4) and VEGF TRAP wherein the plasmids comprise thecoding sequences and either a F2A sequence or an IRES in alternateorientations as described in Example 1. The figures illustrate plasmidscomprising in the 5′ to 3′ direction: sPF4:F2A:VEGF TRAP (FIG. 1A);sPF4: EMCV IRES:VEGF TRAP (FIG. 1B); VEGF TRAP:F2A:sPF4 (FIG. 1C); VEGFTRAP:EMCV IRES:sPF4 (FIG. 1D);

FIGS. 2A-C show expression levels of VEGF-TRAP and sPF4 from plasmidsexpressing both proteins from a single promoter using F2A and IRESsequences, where FIG. 2A illustrates the level of PF4 produced asanalyzed by ELISA (ng/ml); FIG. 2B illustrates the level of VEGF TRAPproduced as analyzed by ELISA (ng/ml); and FIG. 2C illustrates theexpression level of the GFP transfection control indicated as percentpositive.

FIG. 3 depicts an expression cassette for expression of two polypeptides(Factor VIII heavy and light chains) as described in Example 2.

FIG. 4 (SEQ ID NOS: 39-41) depicts an expression cassette for expressionof two polypeptides (Factor VIII heavy and light chains), whereinvarious sequences for B domain deleted forms of Factor VIII arepresented.

FIG. 5 is a schematic illustration of the bioprocessing of Factor VIIIheavy and light chains when they are expressed using a FVIII H2AL (heavychain-2A sequence-light chain) expression cassette comprising a 2Aself-processing cleavage site and an additional proteolytic cleavagesite (Furin).

FIG. 6 is a schematic illustration of an exemplary expression cassettefor expression of monoclonal antibody heavy and light chains where thecassette comprises a 2A self-processing cleavage site, as described inExample 3.

FIG. 7 demonstrates the expression of a full length rat anti-FLK-1monoclonal antibody (IgG) in the supernatant of 293T cells transfectedwith an anti-FLK-1/AAV H2AL (heavy chain-2A sequence-light chain)plasmid.

FIG. 8 demonstrates the biological activity of rat anti-FLK-1 antibody(IgG) expressed in 293T cell supernatants transfected with an anti-FLK-1Ig/AAV H2AL plasmid.

FIGS. 9A and B show the results of Western blot analysis of ratanti-FLK-1 antibody (IgG) in 293T cell supernatants followingtransfection of an anti-FLK-1 Ig/AAV H2AL plasmid. FIG. 9A shows theresults of PAGE using a 12% native gel and FIG. 9B shows the results ofPAGE using a 12% reducing gel wherein Lane 1 shows IgG produced from ahybridoma; Lane 2 shows IgG expressed using a 2A sequence in 293T cellsand Lane 3 is a 293T mock control.

FIG. 10 demonstrates the expression of a full length human anti-KDRmonoclonal antibody (IgG) in the supernatant of 293T cells transfectedwith anti-KDR/AAV H2AL plasmid.

FIG. 11 shows the serum levels of a rat anti-FLK-1 monoclonal antibody(IgG) in mice injected with AAV6H2AL virus.

FIG. 12 depicts an expression cassette encoding an antibody heavy chain,an additional proteolytic cleavage site (Furin), a self-processing 2Acleavage site, and an antibody light chain (HF2AL) for a rat anti-FLK-1antibody as described in Example 6.

FIG. 13 shows expression of a rat anti-FLK-1 antibody in 293T cellsupernatants transfected with a plasmid comprising a sequence encodingan antibody heavy chain and an antibody light chain together with aself-processing 2A cleavage site with and without a furin cleavage site,as described in Example 6.

FIG. 14 shows Western blot characterization of an antibody heavy chainexpressed from 293T (furin +) and LoVo (furin −) cells transfected withH2AL and HF2AL constructs as described in Example 6 and 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides single vector or plasmid constructs forexpression of two or more protein or polypeptide open reading frames andmethods of using the same. The vectors have a self-processing cleavagesequence between the protein or polypeptide coding sequences allowingfor expression of more than one functional protein or polypeptide usinga single promoter. The invention finds utility in production of two ormore proteins or polypeptides or a protein or polypeptide having two ormore domains (or chains) using a single vector where expression occursunder operative control of a single promoter. Exemplary vectorconstructs comprise a self-processing cleavage sequence and may furthercomprise an additional proteolytic cleavage site for removal of theself-processing cleavage sequence from the expressed protein orpolypeptide. The vector constructs find utility in methods relating toenhanced production of biologically active proteins, polypeptides orfragments thereof in vitro and in vivo.

The various compositions and methods of the invention are describedbelow. Although particular compositions and methods are exemplifiedherein, it is understood that any of a number of alternativecompositions and methods are applicable and suitable for use inpracticing the invention. It will also be understood that an evaluationof the protein or polypeptide expression constructs (vectors) andmethods of the invention may be carried out using procedures standard inthe art.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, molecular biology(including recombinant techniques), microbiology, biochemistry andimmunology, which are known to those of skill in the art. Suchtechniques are explained fully in the literature, such as, “MolecularCloning: A Laboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994); and “Current Protocols inImmunology” (J. E. Coligan et al., eds., 1991).

DEFINITIONS

Unless otherwise indicated, all terms used herein have the same meaningas they would to one skilled in the art and the practice of the presentinvention will employ, conventional techniques of microbiology andrecombinant DNA technology, which are within the knowledge of those ofskill of the art.

The term “vector”, as used herein, refers to a DNA or RNA molecule suchas a plasmid, virus or other vehicle, which contains one or moreheterologous or recombinant DNA sequences and is designed for transferbetween different host cells. The terms “expression vector” and “genetherapy vector” refer to any vector that is effective to incorporate andexpress heterologous DNA fragments in a cell. A cloning or expressionvector may comprise additional elements, for example, the expressionvector may have two replication systems, thus allowing it to bemaintained in two organisms, for example in human cells for expressionand in a prokaryotic host for cloning and amplification. Any suitablevector can be employed that is effective for introduction of nucleicacids into cells such that protein or polypeptide expression results,e.g. a viral vector or non-viral plasmid vector. Any cells effective forexpression, e.g., insect cells and eukaryotic cells such as yeast ormammalian cells are useful in practicing the invention.

The terms “heterologous DNA” and “heterologous RNA” refer to nucleotidesthat are not endogenous (native) to the cell or part of the genome inwhich they are present. Generally heterologous DNA or RNA is added to acell by transduction, infection, transfection, transformation or thelike, as further described below. Such nucleotides generally include atleast one coding sequence, but the coding sequence need not beexpressed. The term “heterologous DNA” may refer to a “heterologouscoding sequence” or a “transgene”.

As used herein, the terms “protein” and “polypeptide” may be usedinterchangeably and typically refer to “proteins” and “polypeptides” ofinterest that are expresses using the self processing cleavagesite-containing vectors of the present invention. Such “proteins” and“polypeptides” may be any protein or polypeptide useful for research,diagnostic or therapeutic purposes, as further described below.

The term “replication defective” as used herein relative to a viral genetherapy vector of the invention means the viral vector cannotindependently further replicate and package its genome. For example,when a cell of a subject is infected with rAAV virions, the heterologousgene is expressed in the infected cells, however, due to the fact thatthe infected cells lack AAV rep and cap genes and accessory functiongenes, the rAAV is not able to replicate.

As used herein, a “retroviral transfer vector” refers to an expressionvector that comprises a nucleotide sequence that encodes a transgene andfurther comprises nucleotide sequences necessary for packaging of thevector. Preferably, the retroviral transfer vector also comprises thenecessary sequences for expressing the transgene in cells.

As used herein, “packaging system” refers to a set of viral constructscomprising genes that encode viral proteins involved in packaging arecombinant virus. Typically, the constructs of the packaging systemwill ultimately be incorporated into a packaging cell.

As used herein, a “second generation” lentiviral vector system refers toa lentiviral packaging system that lacks functional accessory genes,such as one from which the accessory genes, vif, vpr, vpu and nef, havebeen deleted or inactivated. See, e.g., Zufferey et al., 1997, Nat.Biotechnol. 15:871-875.

As used herein, a “third generation” lentiviral vector system refers toa lentiviral packaging system that has the characteristics of a secondgeneration vector system, and further lacks a functional tat gene, suchas one from which the tat gene has been deleted or inactivated.Typically, the gene encoding rev is provided on a separate expressionconstruct. See, e.g., Dull et al., 1998, J. Virol. 72(11):8463-8471.

As used herein, “pseudotyped” refers to the replacement of a nativeenvelope protein with a heterologous or functionally modified envelopeprotein.

The term “operably linked” as used herein relative to a recombinant DNAconstruct or vector means nucleotide components of the recombinant DNAconstruct or vector are functionally related to one another foroperative control of a selected coding sequence. Generally, “operablylinked” DNA sequences are contiguous, and, in the case of a secretoryleader, contiguous and in reading frame. However, enhancers do not haveto be contiguous.

As used herein, the term “gene” or “coding sequence” means the nucleicacid sequence which is transcribed (DNA) and translated (mRNA) into apolypeptide in vitro or in vivo when operably linked to appropriateregulatory sequences. The gene may or may not include regions precedingand following the coding region, e.g. 5′ untranslated (5′ UTR) or“leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

A “promoter” is a DNA sequence that directs the binding of RNApolymerase and thereby promotes RNA synthesis, i.e., a minimal sequencesufficient to direct transcription. Promoters and corresponding proteinor polypeptide expression may be cell-type specific, tissue-specific, orspecies specific. Also included in the nucleic acid constructs orvectors of the invention are enhancer sequences which may or may not becontiguous with the promoter sequence. Enhancer sequences influencepromoter-dependent gene expression and may be located in the 5′ or 3′regions of the native gene.

“Enhancers” are cis-acting elements that stimulate or inhibittranscription of adjacent genes. An enhancer that inhibits transcriptionalso is termed a “silencer”. Enhancers can function (i.e., can beassociated with a coding sequence) in either orientation, over distancesof up to several kilobase pairs (kb) from the coding sequence and from aposition downstream of a transcribed region.

A “regulatable promoter” is any promoter whose activity is affected by acis or trans acting factor (e.g., an inducible promoter, such as anexternal signal or agent).

A “constitutive promoter” is any promoter that directs RNA production inmany or all tissue/cell types at most times, e.g., the human CMVimmediate early enhancer/promoter region which promotes constitutiveexpression of cloned DNA inserts in mammalian cells.

The terms “transcriptional regulatory protein”, “transcriptionalregulatory factor” and “transcription factor” are used interchangeablyherein, and refer to a nuclear protein that binds a DNA response elementand thereby transcriptionally regulates the expression of an associatedgene or genes. Transcriptional regulatory proteins generally binddirectly to a DNA response element, however in some cases binding to DNAmay be indirect by way of binding to another protein that in turn bindsto, or is bound to a DNA response element.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene. See, e.g.,Jackson R J, Howell M T, Kaminski A (1990) Trends Biochem Sci15(12):477-83) and Jackson R J and Kaminski, A. (1995) RNA1(10):985-1000. The examples described herein are relevant to the use ofany IRES element, which is able to promote direct internal ribosomeentry to the initiation codon of a cistron. “Under translational controlof an IRES” as used herein means that translation is associated with theIRES and proceeds in a cap-independent manner.

A “self-processing cleavage site” or “self-processing cleavage sequence”is defined herein as a post-translational or co-translational processingcleavage site sequence. Such a “self-processing cleavage” site orsequence refers to a DNA or amino acid sequence, exemplified herein by a2A site, sequence or domain or a 2A-like site, sequence or domain. Asused herein, a “self-processing peptide” is defined herein as thepeptide expression product of the DNA sequence that encodes aself-processing cleavage site or sequence, which upon translation,mediates rapid intramolecular (cis) cleavage of a protein or polypeptidecomprising the self-processing cleavage site to yield discrete matureprotein or polypeptide products.

As used herein, the term “additional proteolytic cleavage site”, refersto a sequence which is incorporated into an expression construct of theinvention adjacent a self-processing cleavage site, such as a 2A or 2Alike sequence, and provides a means to remove additional amino acidsthat remain following cleavage by the self processing cleavage sequence.Exemplary “additional proteolytic cleavage sites” are described hereinand include, but are not limited to, furin cleavage sites with theconsensus sequence RXK(R)R (SEQ ID NO: 10). Such furin cleavage sitescan be cleaved by endogenous subtilisin-like proteases, such as furinand other serine proteases within the protein secretion pathway.

As used herein, the terms “immunoglobulin” and “antibody” may be usedinterchangeably and refer to intact immunoglobulin or antibody moleculesas well as fragments thereof, such as Fa, F(ab′)2, and Fv, which arecapable of binding an antigenic determinant. Such an “immunoglobulin”and “antibody” is composed of two identical light polypeptide chains ofmolecular weight approximately 23,000 daltons, and two identical heavychains of molecular weight 53,000-70,000. The four chains are joined bydisulfide bonds in a “Y” configuration. Heavy chains are classified asgamma (IgG), mu (IgM), alpha (IgA), delta (IgD) or epsilon (IgE) and arethe basis for the class designations of immunoglobulins, whichdetermines the effector function of a given antibody. Light chains areclassified-as either kappa or lambda. When reference is made herein toan “immunoglobulin or fragment thereof”, it will be understood that sucha “fragment thereof” is an immunologically functional immunoglobulinfragment.

The term “humanized antibody” refers to an antibody molecule in whichone or more amino acids of the antigen binding regions of a non-humanantibody have been replaced in order to more closely resemble a humanantibody, while retaining the binding activity of the original non-humanantibody. See, e.g., U.S. Pat. No. 6,602,503.

The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e., an epitope) which makes contact with aparticular antibody. Numerous regions of a protein or fragment of aprotein may induce the production of antibodies which binds specificallyto a given region of the three-dimensional structure of the protein.These regions or structures are referred to as antigenic determinants.An antigenic determinant may compete with the intact antigen (i.e., theimmunogen used to elicit the immune response) for binding to anantibody.

The term “fragment,” when referring to a recombinant protein orpolypeptide of the invention means a polypeptide which has an amino acidsequence which is the same as part of, but not all of, the amino acidsequence of the corresponding full length protein or polypeptide, whichretains at least one of the functions or activities of the correspondingfull length protein or polypeptide. The fragment preferably includes atleast 20-100 contiguous amino acid residues of the full length proteinor polypeptide.

The terms “administering” or “introducing”, as used herein refer todelivery of a vector for recombinant protein expression to a cell or tocells and or organs of a subject. Such administering or introducing maytake place in vivo, in vitro or ex vivo. A vector for recombinantprotein or polypeptide expression may be introduced into a cell bytransfection, which typically means insertion of heterologous DNA into acell by physical means (e.g., calcium phosphate transfection,electroporation, microinjection or lipofection); infection, whichtypically refers to introduction by way of an infectious agent, i.e. avirus; or transduction, which typically means stable infection of a cellwith a virus or the transfer of genetic material from one microorganismto another by way of a viral agent (e.g., a bacteriophage).

“Transformation” is typically used to refer to bacteria comprisingheterologous DNA or cells which express an oncogene and have thereforebeen converted into a continuous growth mode such as tumor cells. Avector used to “transform” a cell may be a plasmid, virus or othervehicle.

Typically, a cell is referred to as “transduced”, “infected”,“transfected” or “transformed” dependent on the means used foradministration, introduction or insertion of heterologous DNA (i.e., thevector) into the cell. The terms “transduced”, “transfected” and“transformed” may be used interchangeably herein regardless of themethod of introduction of heterologous DNA.

As used herein, the terms “stably transformed”, “stably transfected” and“transgenic” refer to cells that have a non-native (heterologous)nucleic acid sequence integrated into the genome. Stable transfection isdemonstrated by the establishment of cell lines or clones comprised of apopulation of daughter cells containing the transfected DNA stablyintegrated into their genomes. In some cases, “transfection” is notstable, i.e., it is transient. In the case of transient transfection,the exogenous or heterologous DNA is expressed, however, the introducedsequence is not integrated into the genome and is considered to beepisomal.

As used herein, “ex vivo administration” refers to a process whereprimary cells are taken from a subject, a vector is administered to thecells to produce transduced, infected or transfected recombinant cellsand the recombinant cells are readministered to the same or a differentsubject.

A “multicistronic transcript” refers to an mRNA molecule that containsmore than one protein coding region, or cistron. A mRNA comprising twocoding regions is denoted a “bicistronic transcript.” The “5′-proximal”coding region or cistron is the coding region whose translationinitiation codon (usually AUG) is closest to the 5′-end of amulticistronic mRNA molecule. A “5′-distal” coding region or cistron isone whose translation initiation codon (usually AUG) is not the closestinitiation codon to the 5′ end of the mRNA. The terms “5′-distal” and“downstream” are used synonymously to refer to coding regions that arenot adjacent to the 5′ end of a mRNA molecule.

As used herein, “co-transcribed” means that two (or more) coding regionsor polynucleotides are under transcriptional control of a singletranscriptional control or regulatory element.

The term “host cell”, as used herein refers to a cell which has beentransduced, infected, transfected or transformed with a vector. Thevector may be a plasmid, a viral particle, a phage, etc. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose skilled in the art. It will be appreciated that the term “hostcell” refers to the original transduced, infected, transfected ortransformed cell and progeny thereof.

As used herein, the terms “biological activity” and “biologicallyactive”, refer to the activity attributed to a particular protein in acell line in culture or in a cell-free system, such as a ligand-receptorassay in ELISA plates. The “biological activity” of an “immunoglobulin”,“antibody” or fragment thereof refers to the ability to bind anantigenic determinant and thereby facilitate immunological function.

As used herein, the terms “tumor” and “cancer” refer to a cell thatexhibits a loss of growth control and forms unusually large clones ofcells. Tumor or cancer cells generally have lost contact inhibition andmay be invasive and/or have the ability to metastasize.

Internal Ribosome Entry Site (IRES)

IRES elements were first discovered in picornavirus mRNAs (Jackson R J,Howell M T, Kaminski A (1990) Trends Biochem Sci 15(12):477-83) andJackson R J and Kaminski, A. (1995) RNA 1(10):985-1000). Examples ofIRES generally employed by those of skill in the art include thosereferenced in Table I, as well as those described in U.S. Pat. No.6,692,736. Examples of “IRES” known in the art include, but are notlimited to IRES obtainable from picornavirus (Jackson et al., 1990) andIRES obtainable from viral or cellular mRNA sources, such as forexample, immunoglobulin heavy-chain binding protein (BiP), the vascularendothelial growth factor (VEGF) (Huez et al. (1998) Mol. Cell. Biol.18(11):6178-6190), the fibroblast growth factor 2 (FGF-2), andinsulin-like growth factor (IGFII), the translational initiation-factoreIF4G and yeast transcription factors TFIID and HAP4, theencephelomycarditis virus (EMCV) which is commercially available fromNovagen (Duke et al. (1992) J. Virol 66(3):1602-9) and the VEGF IRES(Huez et al. (1998) Mol Cell Biol 18(11):6178-90). IRES have also beenreported in different viruses such as cardiovirus, rhinovirus,aphthovirus, HCV, Friend murine leukemia virus (FrMLV) and Moloneymurine leukemia virus (MoMLV). As used herein, “IRES” encompassesfunctional variations of IRES sequences as long as the variation is ableto promote direct internal ribosome entry to the initiation codon of acistron. An IRES may be mammalian, viral or protozoan.

The IRES promotes direct internal ribosome entry to the initiation codonof a downstream cistron, leading to cap-independent translation. Thus,the product of a downstream cistron can be expressed from a bicistronic(or multicistronic) mRNA, without requiring either cleavage of apolyprotein or generation of a monocistronic mRNA. Internal ribosomeentry sites are approximately 450 nucleotides in length and arecharacterized by moderate conservation of primary sequence and strongconservation of secondary structure. The most significant primarysequence feature of the IRES is a pyrimidine-rich site whose start islocated approximately 25 nucleotides upstream of the 3′ end of the IRES.See Jackson et al. (1990).

Three major classes of picornavirus IRES have been identified andcharacterized: (1) the cardio- and aphthovirus class (for example, theencephelomycarditis virus, Jang et al. (1990) Gene Dev 4:1560-1572); (2)the entero- and rhinovirus class (for example, polioviruses, Borman etal. (1994) EMBO J. 13:314903157); and (3) the hepatitis A virus (HAV)class, Glass et al. (1993) Virol 193:842-852). For the first twoclasses, two general principles apply. First, most of the 450-nucleotidesequence of the IRES functions to maintain particular secondary andtertiary structures conducive to ribosome binding and translationalinitiation. Second, the ribosome entry site is an AUG triplet located atthe 3′ end of the IRES, approximately 25 nucleotides downstream of aconserved oligopyrimidine tract. Translation initiation can occur eitherat the ribosome entry site (cardioviruses) or at the next downstream AUG(entero/rhinovirus class). Initiation occurs at both sites inaphthoviruses.

HCV and pestiviruses such as bovine viral diarrhea virus (BVDV) orclassical swine fever virus (CSFV) have 341 nt and 370 nt long 5′-UTRrespectively. These 5′-UTR fragments form similar RNA secondarystructures and can have moderately efficient IRES function(Tsukiyama-Kohara et al. (1992) J. Virol. 66:1476-1483; Frolov I et al.,(1998) RNA 4:1418-1435). Recent studies showed that both Friend-murineleukemia virus (MLV) 5′-UTR and rat retrotransposon virus-like 30S(VL30) sequences contain IRES structure of retroviral origin (Torrent etal. (1996) Hum Gene Ther 7:603-612).

In eukaryotic cells, translation is normally initiated by the ribosomescanning from the capped mRNA 5′ end, under the control of initiationfactors. However, several cellular mRNAs have been found to have IRESstructure to mediate the cap-independent translation (van der Velde, etal. (1999) Int J Biochem Cell Biol. 31:87-106). Examples areimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94), antennapedia mRNA of Drosophilan (Oh et al. (1992)Gene and Dev 6:1643-1653), fibroblast growth factor-2 (FGF-2) (Vagner etal. (1995) Mol Cell Biol 15:3544), platelet-derived growth factor B(PDGF-B) (Bernstein et al. (1997) J Biol Chem 272:9356-9362),insulin-like growth factor 11 (Teerink et al. (1995) Biochim BiophysActa 1264:403-408), and the translation initiation factor eIF4G (Gan etal. (1996) J Biol Chem 271:623-626). Recently, vascular endothelialgrowth factor (VEGF) was also found to have IRES element (Stein et al.(1998) Mol Cell Biol 18:3112-3119; Huez et al. (1998) Mol Cell Biol18:6178-6190).

An IRES sequence may be tested and compared to a 2A sequence as shown inExample 1. In one exemplary protocol a test vector or plasmid isgenerated with one transgene, such as PF-4 or VEGF-TRAP, placed undertranslational control of an IRES, 2A or 2A-like sequence to be tested. Acell is transfected with the vector or plasmid containing the IRES- or2A-reporter gene sequences and an assay is performed to detect thepresence of the transgene. In one illustrative example, the test plasmidcomprises co-transcribed PF-4 and VEGF-TRAP coding sequencestranscriptionally driven by a CMV promoter wherein the PF4 or VEGF-TRAPcoding sequence is translationally driven by the IRES, 2A or 2A-likesequence to be tested. Host cells are transiently transfected with thetest vector or plasmid by means known to those of skill in the art andassayed for the expression of the transgene.

IRES may be prepared using standard recombinant and synthetic methodsknown in the art. For cloning convenience, restriction sites may beengineered into the ends of the IRES fragments to be used.

To express two or more proteins from a single viral or non-viral vector,an internal ribosome entry site (IRES) sequence is commonly used todrive expression of the second, third, fourth gene, etc. Although theuse of an IRES is considered to be the state of the art by many, whentwo genes are linked via an IRES, the expression level of the secondgene is often significantly reduced (Fuder et al., Gene Therapy8:864-873 (2001)). In fact, the use of an IRES to control transcriptionof two or more genes operably linked to the same promoter can result inlower level expression of the second, third, etc. gene relative to thegene adjacent the promoter. In addition, an IRES sequence may besufficiently long to present issues with the packaging limit of thevector, e.g., the eCMV IRES has a length of 507 base pairs.

TABLE 1 LITERATURE REFERENCES FOR IRES IRES Host Example ReferencePicornavirus HAV Glass et al., 1993. Virol 193: 842-852 EMCV Jang &Wimmer, 1990. Gene Dev 4: 1560-1572 Poliovirus Borman et al., 1994. EMBOJ 13: 3149-3157 HCV and HCV Tsukiyama-Kohara et al., 1992. J Virol 66:1476-1483 pestivirus BVDV Frolov I et al., 1998. RNA. 4: 1418-1435Leishmania virus LRV-1 Maga et al., 1995. Mol Cell Biol 15: 4884-4889Retroviruses MoMLV Torrent et al., 1996. Hum Gene Ther 7: 603-612 VL30(Harvey murine sarcoma virus) REV Lopez-Lastra et al., 1997. Hum GeneTher 8: 1855-1865 Eukaryotic BiP Macejak & Sarnow, 1991. Nature 353:90-94 mRNA antennapedia Oh et al., 1992. Gene & Dev 6: 1643-1653 mRNAFGF-2 Vagner et al., 1995. Mol Cell Biol 15: 35-44 PDGF-B Bernstein etal., 1997. J Biol Chem 272: 9356-9362 IGFII Teerink et al., 1995.Biochim Biophys Acta 1264: 403-408 eIF4G Gan & Rhoads, 1996. J Biol Chem271: 623-626 VEGF Stein et al., 1998. Mol Cell Biol 18: 3112-3119; Huezet al., 1998. Mol Cell Biol 18: 6178-6190

The linking of proteins in the form of polyproteins is a strategyadopted in the replication of many viruses including picornaviridae.Upon translation, virus-encoded self-processing peptides mediate rapidintramolecular (cis) cleavage of the polyprotein to yield discretemature protein products. The present invention provides advantages overthe use of an IRES in that a vector for recombinant protein orpolypeptide expression comprising a self-processing peptide (exemplifiedherein by 2A peptides) is provided which facilitates expression of twoor more protein or polypeptide coding sequences using a single promoter,wherein the two or more proteins or polypeptides are expressed in asubstantially equimolar ratio.

Self-Processing Cleavage Sites or Sequences

A “self-processing cleavage site” or “self-processing cleavage sequence”as defined above refers to a DNA or amino acid sequence, wherein upontranslation, rapid intramolecular (cis) cleavage of a polypeptidecomprising the self-processing cleavage site occurs to result inexpression of discrete mature protein or polypeptide products. Such a“self-processing cleavage site”, may also be referred to as apost-translational or co-translational processing cleavage site,exemplified herein by a 2A site, sequence or domain. It has beenreported that a 2A site, sequence or domain demonstrates a translationaleffect by modifying the activity of the ribosome to promote hydrolysisof an ester linkage, thereby releasing the polypeptide from thetranslational complex in a manner that allows the synthesis of adiscrete downstream translation product to proceed (Donnelly, 2001).Alternatively, a 2A site, sequence or domain demonstrates“auto-proteolysis” or “cleavage” by cleaving its own C-terminus in cisto produce primary cleavage products (Furler; Palmenberg, Ann. Rev.Microbiol. 44:603-623 (1990)).

Although the mechanism is not part of the invention, the activity of a2A-like sequence may involve ribosomal skipping between codons whichprevents formation of peptide bonds (de Felipe et al., Human GeneTherapy 11:1921-1931 (2000); Donnelly et al., J. Gen. Virol.82:1013-1025 (2001)), although it has been considered that the domainacts more like an autolytic enzyme (Ryan et al., Virol. 173:35-45(1989). Studies in which the Foot and Mouth Disease Virus (FMDV) 2Acoding region was cloned into expression vectors and transfected intotarget cells showed FMDV 2A cleavage of artificial reporter polyproteinsin wheat-germ lysate and transgenic tobacco plants (Halpin et al., U.S.Pat. No. 5,846,767; 1998 and Halpin et al., The Plant Journal17:453-459, 1999); Hs 683 human glioma cell line (de Felipe et al., GeneTherapy 6:198-208, 1999); hereinafter referred to as “de Felipe II”);rabbit reticulocyte lysate and human HTK-143 cells (Ryan et al., EMBO J.13:928-933 (1994)); and insect cells (Roosien et al., J. Gen. Virol.71:1703-1711, 1990). The FMDV 2A-mediated cleavage of a heterologouspolyprotein has been shown for IL-12 (p40/p35 heterodimer; Chaplin etal., J. Interferon Cytokine Res. 19:235-241, 1999). The referencedemonstrates that in transfected COS-7 cells, FMDV 2A mediated thecleavage of a p40-2A-p35 polyprotein into biologically functionalsubunits p40 and p35 having activities associated with IL-12.

The FMDV 2A sequence has been incorporated into retroviral vectors,alone or combined with different IRES sequences to constructbicistronic, tricistronic and tetracistronic vectors. The efficiency of2A-mediated gene expression in animals was demonstrated by Furler (2001)using recombinant adeno-associated viral (AAV) vectors encodingα-synuclein and EGFP or Cu/Zn superoxide dismutase (SOD-1) and EGFPlinked via the FMDV 2A sequence. EGFP and α-synuclein were expressed atsubstantially higher levels from vectors which included a 2A sequencerelative to corresponding IRES-based vectors, while SOD-1 was expressedat comparable or slightly higher levels. Fuder also demonstrated thatthe 2A sequence results in bicistronic gene expression in vivo afterinjection of 2A-containing AAV vectors into rat substantia nigra.

For the present invention, the DNA sequence encoding a self-processingcleavage site is exemplified by viral sequences derived from apicornavirus, including but not limited to an entero-, rhino-, cardio-,aphtho- or Foot-and-Mouth Disease Virus (FMDV). In a preferredembodiment, the self-processing cleavage site coding sequence is derivedfrom a FMDV. Self-processing cleavage sites include but are not limitedto 2A and 2A-like sites, sequences or domains (Donnelly et al., J. Gen.Virol. 82:1027-1041 (2001).

Positional subcloning of a 2A sequence between two or more heterologousDNA sequences for the inventive vector construct allows the delivery andexpression of two or more open reading frames by operable linkage to asingle promoter. Preferably, self processing cleavage sites such as FMDV2A sequences provide a unique means to express and deliver from a singleviral vector, two or more proteins, polypeptides or peptides which canbe individual parts of, for example, Factor VIII or anotherheterodimeric protein, an antibody, or a heterodimeric receptor.

FMDV 2A is a polyprotein region which functions in the FMDV genome todirect a single cleavage at its own C-terminus, thus functioning in cis.The FMDV 2A domain is typically reported to be about nineteen aminoacids in length ((LLNFDLLKLAGDVESNPGP (SEQ ID NO: 1);TLNFDLLKLAGDVESNPGP (SEQ ID NO: 2); Ryan et al., J. Gen. Virol.72:2727-2732 (1991)), however oligopeptides of as few as fourteen aminoacid residues ((LLKLAGDVESNPGP (SEQ ID NO: 3)) have also been shown tomediate cleavage at the 2A C-terminus in a fashion similar to its rolein the native FMDV polyprotein processing.

Variations of the 2A sequence have been studied for their ability tomediate efficient processing of polyproteins (Donnelly M L L et al.2001). Homologues and variant 2A sequences are included within the scopeof the invention and include but are not limited to the sequencespresented in Table 2, below:

TABLE 2 Table of Exemplary 2A Sequences LLNFDLLKLAGDVESNPGP (SEQ IDNO: 1) TLNFDLLKLAGDVESNPGP; (SEQ ID NO: 2) LLKLAGDVESNPGP (SEQ ID NO: 3)NFDLLKLAGDVESNPGP (SEQ ID NO: 4) QLLNFDLLKLAGDVESNPGP (SEQ ID NO: 5)APVKQTLNFDLLKLAGDVESNPGP. (SEQ ID NO: 6)VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQTLNFDLLKLA GDVESNPGP (SEQ ID NO:7) LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 8)EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 9)

Distinct advantages of self-processing cleavage sequences, such as a 2Asequence or a variant thereof are their use in generating vectorsexpressing self-processing polyproteins. This invention includes anyvector (plasmid or viral based) which comprises the coding sequence fortwo or more proteins or polypeptides linked via self-processing cleavagesites such that the individual proteins or polypeptides are expressed inequimolar or close to equimolar amounts following the cleavage of thepolyprotein due to the presence of the self-processing cleavage site.These proteins may be heterologous to the vector itself, to each otheror to the self-processing cleavage site, e.g., FMDV. Thus theself-processing cleavage sites for use in practicing the invention donot discriminate between heterologous proteins or polypeptides andcoding sequences derived from the same source as the self-processingcleavage site, in the ability to function or mediate cleavage.

The small size of the 2A coding sequence further enables its use invectors with a limited packing capacity for a coding sequence such asAAV. The utility of AAV vectors can be further expanded since the 2Asequence eliminates the need for dual promoters. The expression levelsof individual proteins, polypeptides or peptides from a promoter drivinga single open reading frame comprising more than two coding sequencesare closer to equimolar as compared to expression levels achievableusing IRES sequences or dual promoters. Elimination of dual promotersreduces promoter interference that may result in reduced and/or impairedlevels of expression for each coding sequence.

In one preferred embodiment, the FMDV 2A sequence included in a vectoraccording to the invention encodes amino acid residues comprisingLLNFDLLKLAGDVESNPGP (SEQ ID NO:1). Alternatively, a vector according tothe invention may encode amino acid residues for other 2A-like regionsas discussed in Donnelly et al., J. Gen. Virol. 82:1027-1041 (2001) andincluding but not limited to a 2A-like domain from picornavirus, insectvirus, Type C rotavirus, trypanosome repeated sequences or thebacterium, Thermatoga maritima.

The invention contemplates the use of nucleic acid sequence variantsthat encode a self-processing cleavage site, such as a 2A or 2A-likepolypeptide, and nucleic acid coding sequences that have a differentcodon for one or more of the amino acids relative to that of the parent(native) nucleotide. Such variants are specifically contemplated andencompassed by the present invention. Sequence variants ofself-processing cleavage peptides and polypeptides are included withinthe scope of the invention as well.

As used herein, the term “sequence identity” means nucleic acid or aminoacid sequence identity between two or more aligned sequences, whenaligned using a sequence alignment program. The terms “% homology” and“% identity” are used interchangeably herein and refer to the level ofnucleic acid or amino acid sequence identity between two or more alignedsequences, when aligned using a sequence alignment program. For example,80% homology means the same thing as 80% sequence identity determined bya defined algorithm under defined conditions.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch,J. Mol. Biol. 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), by the BLAST algorithm, Altschulet al., J. Mol. Biol. 215: 403-410 (1990), with software that ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/), or by visual inspection (seegenerally, Ausubel et al., infra). For purposes of the presentinvention, optimal alignment of sequences for comparison is mostpreferably conducted by the local homology algorithm of Smith &Waterman, Adv. Appl. Math. 2: 482 (1981). See, also, Altschul, S. F. etal., 1990 and Altschul, S. F. et al., 1997.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms described herein, e.g. the Smith-Watermanalgorithm, or by visual inspection.

In accordance with the present invention, also encompassed are sequencevariants which encode self-processing cleavage polypeptides andpolypeptides themselves that have 80, 85, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% or more sequence identity to the native sequence.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm-5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “intermediate stringency” at about 10-20° below the Tm of the probe;and “low stringency” at about 20-25° below the Tm. Functionally, maximumstringency conditions may be used to identify sequences having strictidentity or near-strict identity with the hybridization probe; whilehigh stringency conditions are used to identify sequences having about80% or more sequence identity with the probe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, andin Ausubel, F. M., et al., 1993. An example of high stringencyconditions includes hybridization at about 42° C. in 50% formamide,5×SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrierDNA followed by washing two times in 2×SSC and 0.5% SDS at roomtemperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C.2A sequence variants that encode a polypeptide with the same biologicalactivity as the 2A polypeptides described herein and hybridize undermoderate to high stringency hybridization conditions are considered tobe within the scope of the present invention.

As a result of the degeneracy of the genetic code, a number of codingsequences can be provided which encode the same protein, polypeptide orpeptide, such as 2A or a 2A-like peptide. For example, the triplet CGTencodes the amino acid arginine. Arginine is alternatively encoded byCGA, CGC, CGG, AGA, and AGG. Therefore it is appreciated that suchsubstitutions in the coding region fall within the sequence variantsthat are covered by the present invention.

It is further appreciated that such sequence variants may or may nothybridize to the parent sequence under conditions of high stringency.This would be possible, for example, when the sequence variant includesa different codon for each of the amino acids encoded by the parentnucleotide. Such variants are, nonetheless, specifically contemplatedand encompassed by the present invention.

Removal of Self-Processing Peptide Sequences.

One concern associated with the use of self-processing peptides, such asa 2A or 2A-like sequence is that the C terminus of the expressedpolypeptide contains amino acids derived from the self-processingpeptide, i.e. 2A-derived amino acid residues. These amino acid residuesare “foreign” to the host and may elicit an immune response when therecombinant protein is expressed in vivo (i.e., expressed from a viralor non-viral vector in the context of gene therapy or administered as anin vitro-produced recombinant protein or polypeptide) or delivered invivo following in vitro or ex vivo expression. In addition, if notremoved, self-processing peptide-derived amino acid residues mayinterfere with protein secretion in producer cells and/or alter proteinconformation, resulting in a less than optimal expression level and/orreduced biological activity of the recombinant protein.

The invention includes expression constructs, engineered such that anadditional proteolytic cleavage site is provided between a first proteinor polypeptide coding sequence (the first or 5′ ORF) and the selfprocessing cleavage site as a means for removal of self processingcleavage site derived amino acid residues that are present in theexpressed protein product.

Examples of additional proteolytic cleavage sites are furin cleavagesites with the consensus sequence RXK(R)R (SEQ ID NO: 10), which can becleaved by endogenous subtilisin-like proteases, such as furin and otherserine proteases. As shown in Example 6, the inventors have demonstratedthat self processing 2A amino acid residues at the C terminus of a firstexpressed protein can be efficiently removed by introducing a furincleavage site RAKR (SEQ ID NO: 38) between the first polypeptide and aself processing 2A sequence. In addition, use of a plasmid containing a2A sequence and a furin cleavage site adjacent to the 2A sequence wasshown to result in a higher level of protein expression than a plasmidcontaining the 2A sequence alone. This improvement provides a furtheradvantage in that when 2A amino acid residues are removed from theC-terminus of the protein, longer 2A- or 2A like sequences or otherself-processing sequences can be used.

It is often advantageous to produce therapeutic proteins, polypeptides,fragments or analogues thereof with fully human characteristics. Thesereagents avoid the undesired immune responses induced by proteins,polypeptides, fragments or analogues thereof originating from differentspecies. To address possible host immune responses to amino acidresidues derived from self-processing peptides, the coding sequence fora proteolytic cleavage site may be inserted (using standard methodologyknown in the art) between the coding sequence for a first protein andthe coding sequence for a self-processing peptide so as to remove theself-processing peptide sequence from the expressed protein orpolypeptide. This finds particular utility in therapeutic and diagnosticproteins and polypeptides for use in vivo.

Any additional proteolytic cleavage site known in the art that can beexpressed using recombinant DNA technology may be employed in practicingthe invention. Exemplary additional proteolytic cleavage sites which canbe inserted between a polypeptide or protein coding sequence and a selfprocessing cleavage sequence include, but are not limited to a:

a). Furin consensus sequence or site: RXK(R)R (SEQ ID. NO:10);

b). Factor Xa cleavage sequence or site: IE(D)GR (SEQ ID. NO:11);

c). Signal peptidase I cleavage sequence or site: e.g., LAGFATVAQA (SEQID. NO:12); and

d). Thrombin cleavage sequence or site: LVPRGS (SEQ ID. NO:13).

Protein Coding Sequences

As used herein, a “first protein coding sequence” refers to aheterologous nucleic acid sequence encoding a polypeptide or proteinmolecule or domain or chain thereof including, but not limited to achain of an antibody or immunoglobulin molecule or fragment thereof, acytokine or fragment thereof, a growth factor or fragment thereof, achain of a Factor VIII molecule, a soluble or membrane-associatedreceptor or fragment thereof, a viral protein or fragment thereof, animmunogenic protein or fragment thereof, a transcriptional regulator orfragment thereof, a proapoptotic molecule or fragment thereof, a tumorsuppressor or fragment thereof, an angiogenesis inhibitor or fragmentthereof, etc.

As used herein, a “second protein coding sequence” refers to aheterologous nucleic acid sequence encoding: a polypeptide or proteinmolecule or domain or chain thereof including, but not limited to achain of an antibody or immunoglobulin or fragment thereof, a cytokineor fragment thereof, a growth factor or fragment thereof, a chain of aFactor VIII molecule, a soluble or membrane-associated receptor orfragment thereof, a viral protein or fragment thereof, an immunogenicprotein or fragment thereof, a transcriptional regulator or fragmentthereof, a proapoptotic molecule or fragment thereof, a tumor suppressoror fragment thereof, an angiogenesis inhibitor or fragment thereof, etc.

The vector constructs of the invention may comprise two or moretransgenes or heterologous coding sequences, e.g., a first proteincoding sequence, a second protein coding sequence, a third proteincoding sequence, etc. Numerous transgenes may be employed in thepractice of the present invention and include, but are not limited to,nucleotide sequences encoding one or more of the proteins indicatedbelow or a fragment thereof:

1. A sequence encoding HIF-1α and HIFβ (HIF), p35 and p40 (IL-12), chainA and chain B of insulin, integrins such as, but not limited to alpha Vbeta 3 or alpha V beta 5, antibody heavy and light chains and the heavyand light chain of Factor VIII.

2. A sequence encoding a soluble receptor, include but are not limitedto, the TNF p55 and p75 receptor, the IL-2 receptor, the FGF receptors,the VEGF receptors, TIE2, the IL-6 receptor and the IL-1 receptor;

3. A sequence encoding a cytokine including, but not limited to, anyknown or later discovered cytokine, for example, IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, 11-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-18, IL-24,INF-α, INF-β, INF-γ, GM-CSF, G-CSF and erythropoietin.

4. A sequence encoding a growth factor including, but not limited to,VEGF, FGF, Angiopoietin-1 and 2, PDGF, EGF, IGF, NGF, IDF, HGF, TGF-α,TGF-beta.

5. A sequence encoding a pro-apoptotic factor including, but not limitedto, Bad, Bak, Bax, Bcl2, Bcl-Xs, Bik, Caspases, FasL, and TRAIL.

6. A sequence encoding a tumor suppressor protein or cell cycleregulator including, but not limited to, p53, p16, p19, -21, p27, PTEN,RB1.

7. A sequence encoding an angiogenesis regulator including, but notlimited to, angiostatin, endostatin, TIMPs, antithrombin, plateletfactor 4 (PF4), soluble forms of VEGFR1 (domains 1-7) and VEGFR2(domains 1-7) fused to an Fc segment of IgG1, VEGF-TRAP, PEDF, PEX,troponin 1, thrombospondin, tumstatin, 16 Kd Prolactin.

Cloned sequences and full length nucleotides encoding any of theabove-referenced biologically active molecules may be obtained by wellknown methods in the art (Sambrook et al., 1989). In general, thenucleic acid coding sequences are known and may be obtained from publicdatabases and/or scientific publications.

Homologues and variants of heterologous protein and polypeptide codingsequences are included within the scope of the invention based on“sequence identity” or “% homology” to known nucleic acid sequenceswhich are available in public databases and/or selective hybridizationunder stringent conditions to such known nucleic acid sequences, asdescribed above for self processing cleavage sequences. Homologues andvariants of heterologous protein and polypeptide amino acid sequencesand nucleic acid sequences that encode them are further included withinthe scope of the invention. Such sequences may be identified based on“sequence identity” to known sequences using publicly availabledatabases and sequence alignment programs, as described above for selfprocessing cleavage sequences.

Protein Expression

High level expression of therapeutic proteins has been successfullydemonstrated in the milk of transgenic goats. Taking monoclonalantibodies as an example, it has been shown that antigen binding levelsare equivalent to that of monoclonal antibodies produced usingconventional cell culture technology. This method is based ondevelopment of human therapeutic proteins in the milk of transgenicanimals, which carry genetic information allowing them to express humantherapeutic proteins in their milk. Once they are produced, theserecombinant proteins can be efficiently purified from milk usingstandard technology. See e.g., Pollock, D. P. et al., Journal ofImmunological Methods. 231: 147-157, 1999 and Young, M. W. et al., ResImmunol. July-August; 149(6): 609-610, 1998. Animal milk, egg white,blood, urine, seminal plasma and silk worm cocoons from transgenicanimals have demonstrated potential as sources for production ofrecombinant proteins at an industrial scale (Houdebine L M, Curr OpinBiotechnology, 13: 625-629, 2002; Little M et al., Immunol Today,21(8):364-70, 2000; and Gura T, Nature, 417:584-586, 2002). Theinvention contemplates use of transgenic animal expression systems forexpression of a recombinant protein or polypeptide using theself-processing cleavage site-encoding vectors of the invention.

Production of recombinant proteins in plants has also been successfullydemonstrated including, but not limited to rice transformed byAgrobacterium infection, recombinant human GM-CSF expression in theseeds of transgenic tobacco plants and expression of single-chainantibodies in plants. See, e.g., Streatfield S J, Howard J A, Int J.Parasitol. 33(5-6):479-93, 2003; Schillberg S. et al., Cell Mol LifeSci. 60(3):433-45, 2003; Pogue G P et al., Annu Rev Phytopathol.40:45-74, 2002; and McCormick M et al., J Immunological Methods,278(1-2):95-104, 2003. The invention contemplates use of transgenicplant expression systems for expression of a recombinant protein orpolypeptide using the self-processing cleavage site-encoding vectors ofthe invention.

Baculovirus vector expression systems in conjunction with insect cellsare also gaining ground as a viable platform for recombinant proteinproduction. Baculovirus vector expression systems have been reported toprovide advantages relative to mammalian cell culture expression systemssuch as ease of culture and higher expression levels. See, e.g., GhoshS. et al., Mol Ther. 2002 July; 6(1):5-11, 2002 and Ikonomou L et al.,Appl Microbiol Biotechnol. 62(1):1-20, 2003. The invention furthercontemplates use of Baculovirus vector expression systems for expressionof a recombinant protein or polypeptide using the self-processingcleavage site-encoding vectors of the invention.

Yeast-based systems may also be employed for expression of a recombinantprotein or polypeptide using the self-processing cleavage site-encodingvectors of the invention. See, e.g., Stuart, WD (1997): “Heterologousdimeric proteins produced in heterokaryons”; U.S. Pat. No. 5,643,745.

It will be understood that the vectors of the invention which comprisethe coding sequence for a self-processing peptide alone or incombination with an additional coding sequence for a proteolyticcleavage site find utility in the expression of recombinant proteins andpolypeptides in any protein expression system, a number of which areknown in the art and examples of which are described herein. One ofskill in the art may easily adapt the vectors of the invention for usein any protein expression system.

Following expression, recombinant proteins are recovered from theculture using standard techniques known in the art. The production andrecovery of recombinant proteins themselves can be achieved in variousways numerous examples of which are known in the art. For example, theproduction of a recombinant protein, polypeptide, an analogue orfragment thereof, can be undertaken by culturing the modifiedrecombinant host cell under culture conditions appropriate that hostcell resulting in expression of the coding sequence(s). In order tomonitor the success of expression, recombinant protein or polypeptidelevels are monitored using standard techniques such as ELISA, RIA,Western blot and the like.

Purified forms of the recombinant proteins can, of course, be readilyprepared by standard purification techniques known in the art, e.g.,affinity chromatography. Recombinant proteins can also be purified usingconventional chromatography, such as an ion exchange or size exclusioncolumn, in conjunction with other technologies, such as size-limitedmembrane filtration. The expression systems are preferably designed toinclude signal peptides so that the resulting recombinant proteins aresecreted into the medium, however, intracellular production is alsopossible.

The operability of the present invention has been further demonstratedby expression of platelet factor 4 (PF4) and VEGF-TRAP using the selfprocessing cleavage sequence-containing vectors of the present invention(Example 1). The advantages associated with use of self-processingcleavage sequences are enhanced by inclusion of an additionalproteolytic cleavage site between the coding sequence for a firstprotein or polypeptide and the self-processing cleavage sequence in thevectors of the invention, resulting in removal of amino acid residuesassociated with the self-processing cleavage sequence. Efficient removalof 2A residues by incorporation of a furin cleavage site in the vectorsof the invention is demonstrated in Examples 6 and 7.

A. Platelet Factor 4 (PF4) and VEGF-TRAP

Platelet factor 4 (PF-4) is a member of the CXC family of chemokines andhas been shown to be a potent in vitro inhibitor of endothelial cellproliferation and an in vivo inhibitor of angiogenesis (Maione, T E etal. Science 237:77-79, 1990). Furthermore, recombinant PF-4 has beenshown to inhibit the growth of B16F10 melanoma and HCT colon carcinomacells (Sharpe, R J et al. J. Natl. Cancer Inst. 82:848-853, 1990, Kolberet al. J. Natl. Cancer Inst. 87:304-309, 1995). Adenoviral or retroviraldelivery of a secreted form of PF-4 (sPF-4) was further shown to inhibitthe growth of rat RT2 and human U87MG glioma cells through anangiogenesis-dependent mechanism (Tanaka et al., Nat. Med. 3:437-442,1997). PF-4 appears to block angiogenesis by interfering with thebinding of FGF-2 and VEGF binding to their receptors (Perollet, C. Blood91:3289-3299, 1998, Gengrinovitch et al, J. Biol. Chem. 270:15059-15065,1995).

VEGF-Trap consists of the signal sequence and domain 2 of VEGFR1attached to domain 3 of VEGFR2 and a human IgG Fc region (Holash et al.Proc. Natl. Acad. Sci. USA. 99(17):11393-8, 2002;). Each of the domainswas PCR amplified separately using the oligonucleotides shown below, andthe resulting products were fused by PCR SOEing to generate the finalnucleotide sequence. The nucleotide sequences for VEGFR1 and VEGF-R2were obtained from the plasmids pBLAST-hFLT1 (Invivogen) andpBLAST-hFLK1 (Invivogen).

B. Factor VIII

Hemophilia A is an X-linked recessive bleeding disorder characterized bya deficiency or functional defect in the coagulation Factor VIII. Thereare approximately 20,000 Hemophilia A patients in the United States, andthe cost of treatment for severely affected individuals approaches100,000 per year. Without Factor VIII, patients can experiencelife-threatening blood loss from minor scrapes and cuts. The severity ofsymptoms associated with hemophilia is related to the amount of theclotting factor in the blood. If the level of circulating Factor VIII inhemophilia patients is increased to five percent of the normal level ofFactor VIII, symptoms are mild, with rare bleeding except after injuriesor surgery. The most important challenges facing the hemophilia patientare the availability, cost, and safety of products used for treatment.Plasma derived Factor VIII concentrates were extensively used in the1970s and 1980s. Unfortunately, these concentrates carried a significantrisk of viral contamination. Factor VIII is now available in severaldifferent recombinant forms, however therapy is limited by theavailability, in vivo half-life, and the high cost of treatment. Therecombinant Factor VIII products include a natural full-lengthrecombinant Factor VIII form, and a B-domain deleted recombinant FactorVIII form.

In plasma, Factor VIII exists as a metal ion heterodimer of a variablysized 90-200 kDa heavy chain and 80 kDa light chain. The mature FactorVIII protein contains 2332 amino acids arranged in six domains, namelyA1 (residues 1-336), A2 (372-710), B (741-1648), A3 (1896-2019), C1(2020-2172), C2 (2173-2332). The heavy chain encodes the A1, A2, and Bdomains, whereas the light chain encodes the A3, C1, and C2 domains. TheFactor VIII protein is highly glycosylated and contains 25 consensussites for N-linked glycosylation, 19 of which are located in theB-domain. The B-domain is proteolytically released upon activation bythrombin and is not required for Factor VIII procoagulant activity invitro or in vivo (Lenting, P. J., et al (1998) Blood 92 11, 3983-3996).A number of B-domain deleted forms of Factor VIII have been generated.Common Factor VIII constructs include those where the B-domain iscompletely removed or replaced with 1-4 Arg residues (Lind, P., et al.Eur J Biochem 232 1, 19-27, 1995). Removal of the B-domain has noadverse effect on protein activity, and results in an approximately20-fold increase in level of Factor VIII (Pittman et al., Blood81:2925-2935, 1993).

Gene therapy was thought to offer the promise of a new method oftreating Hemophilia A. However, although numerous reports of Factor VIIIexpression using gene delivery technology may be found in the scientificliterature, production of therapeutic levels of the protein remains achallenge (Vandendriessche et al., J. Thromb. Haemost. 1:1550-1558,2003).

The present invention provides improved vectors and methods for theexpression of Factor VIII protein or a functional variant thereof. Themethod comprises the stable introduction of a nucleic acid constructencoding the Factor VIII polypeptide or a functional variant thereof anda self-processing cleavage site into a cell in vivo, in vitro or exvivo. The nucleic acid sequence encoding Factor VIII may contain genomicor complementary DNA. There are many applications for this method,including the manufacturing of recombinant Factor VIII used in theprophylactic and acute treatment of Hemophilia A. Other applicationsinclude the in vivo and ex vivo delivery of Factor VIII to patients,e.g., using AAV or lentiviral vectors. An example of an AAV vectorcontaining the Factor VIII gene is shown in FIG. 3.

It is not intended that the present invention be limited to any specificFactor VIII sequence or gene delivery mechanism. Many natural andrecombinant forms of Factor VIII have been identified and characterized.Therefore, included within the scope of the invention are any known, orlater discovered, DNA sequences coding for biologically active FactorVIII that can be expressed using the vectors and methods of the presentinvention. Examples of naturally occurring and recombinant forms ofFactor VIII can be found in the patent and scientific literatureincluding, but not limited to High K A, Semin Thromb Hemost. 2003February; 29(1):107-20; Thompson A R. Semin Thromb Hemost. 2003February; 29(1):11-22; Sandberg H et al., Semin Hematol. 2001 April;38(2 Suppl 4):4-12; Brinkhous K et al., Semin Thromb Hemost. 2002 June;28(3):269-72; Osterberg T et al., Semin Hematol. 2001 April; 38(2 Suppl4):40-3; Kjalke M et al., Eur J. Biochem. 1995 Dec. 15; 234(3):773-9;Lind P et al., Eur J Biochem. 1995 Aug. 15; 232(1):19-27; Sanberg etal., XXth Int. Congress of the World Fed. Of Hemophilia (1992); U.S.Pat. Nos. 6,649,375; 6,649,375; 6,642,028; 6,599,724; 6,518,482;6,517,830; 6,458,563; 6,376,463; 6,358,703; 6,358,236; 6,320,029;6,271,025; 6,251,632; 6,221,349; 6,200,560; 6,180,371; PCT PublicationNos. WO 03/100053; WO 03/087161; WO 03/080108; WO 03/047507; WO03/031598; WO 02/072023; WO 02/24723; WO 01/68109; WO 01/45510; WO01/27303; WO 01/03726; WO 00/71141; WO 00/23116; WO 99/61642; WO99/61595; WO 99/46299; WO 99/46274; and WO 97/49725.

Homologues and variants of Factor VIII nucleic acid and amino acidsequences are included within the scope of the invention based on“sequence identity” or “% homology” to known nucleic acid sequenceswhich are available in public databases and/or selective hybridizationunder stringent conditions in the case of nucleic acid sequences, asdescribed above for self processing cleavage sequences.

It will be understood that the vectors of the invention which comprisethe coding sequence for a self-processing peptide alone or incombination with an additional proteolytic cleavage site find utility inthe expression of recombinant Factor VIII in any protein expressionsystem, a number of which are known in the art and examples of which aredescribed herein.

Recombinant Factor VIII is recovered from the culture medium ifexpressed in vitro, or from plasma or other body fluids if expressed invivo using standard techniques routinely used by those of skill in theart. Methods such as immunoassay (e.g., ELISA) and coagulation assaysare typically employed in evaluating the production of Factor VIII andthe biological activity thereof, however, it is not intended that thepresent invention be limited to any particular method of evaluation.

C. Immunoglobulins and Fragments Thereof

Antibodies are immunoglobulin proteins that are heterodimers of a heavyand light chain and have proven difficult to express in a full lengthform from a single vector in mammalian culture expression systems. Threemethods are currently used for production of vertebrate antibodies, invivo immunization of animals to produce “polyclonal” antibodies, invitro cell culture of B-cell hybridomas to produce monoclonal antibodies(Kohler, et al., Eur. J. Immunol., 6: 511, 1976; Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988) and recombinantDNA technology (described for example in Cabilly et al., U.S. Pat. No.6,331,415).

The basic molecular structure of immunoglobulin polypeptides is known toinclude two identical light chains with a molecular weight ofapproximately 23,000 daltons, and two identical heavy chains with amolecular weight 53,000-70,000, where the four chains are joined bydisulfide bonds in a “Y” configuration. The amino acid sequence runsfrom the N-terminal end at the top of the Y to the C-terminal end at thebottom of each chain. At the N-terminal end is a variable region (ofapproximately 100 amino acids in length) which provides for thespecificity of antigen binding.

The present invention provides improved methods for production ofimmunoglobulins of all types, including, but not limited to full lengthantibodies and antibody fragments having a native sequence (i.e. thatsequence produced in response to stimulation by an antigen), singlechain antibodies which combine the antigen binding variable region ofboth the heavy and light chains in a single stably-folded polypeptidechain; univalent antibodies (which comprise a heavy chain/light chaindimer bound to the Fc region of a second heavy chain); “Fab fragments”which include the full “Y” region of the immunoglobulin molecule, i.e.,the branches of the “Y”, either the light chain or heavy chain alone, orportions, thereof (i.e., aggregates of one heavy and one light chain,commonly known as Fab′); “hybrid immunoglobulins” which have specificityfor two or more different antigens (e.g., quadromas or bispecificantibodies as described for example in U.S. Pat. No. 6,623,940);“composite immunoglobulins” wherein the heavy and light chains mimicthose from different species or specificities; and “chimeric antibodies”wherein portions of each of the amino acid sequences of the heavy andlight chain are derived from more than one species (i.e., the variableregion is derived from one source such as a murine antibody, while theconstant region is derived from another, such as a human antibody).

The compositions and methods of the invention find utility in productionof immunoglobulins or fragments thereof wherein the heavy or light chainis “mammalian”, “chimeric” or modified in a manner to enhance itsefficacy. Modified antibodies include both amino acid and nucleic acidsequence variants which retain the same biological activity of theunmodified form and those which are modified such that the activity isaltered, i.e., changes in the constant region that improve complementfixation, interaction with membranes, and other effector functions, orchanges in the variable region that improve antigen bindingcharacteristics. The compositions and methods of the invention furtherinclude catalytic immunoglobulins or fragments thereof.

A “variant” immunoglobulin-encoding polynucleotide sequence may encode a“variant” immunoglobulin amino acid sequence which is altered by one ormore amino acids from the reference polypeptide sequence. The variantpolynucleotide sequence may encode a variant amino acid sequence whichcontains “conservative” substitutions, wherein the substituted aminoacid has structural or chemical properties similar to the amino acidwhich it replaces. In addition, or alternatively, the variantpolynucleotide sequence may encode a variant amino acid sequence whichcontains “non-conservative” substitutions, wherein the substituted aminoacid has dissimilar structural or chemical properties to the amino acidwhich it replaces. Variant immunoglobulin-encoding polynucleotides mayalso encode variant amino acid sequences which contain amino acidinsertions or deletions, or both. Furthermore, a variant“immunoglobulin-encoding polynucleotide” may encode the same polypeptideas the reference polynucleotide sequence but, due to the degeneracy ofthe genetic code, has a polynucleotide sequence altered by one or morebases from the reference polynucleotide sequence.

The term “fragment,” when referring to a recombinant immunoglobulin ofthe invention means a polypeptide which has an amino acid sequence whichis the same as part of but not all of the amino acid sequence of thecorresponding full length immunoglobulin protein, which either retainsessentially the same biological function or activity as thecorresponding full length protein, or retains at least one of thefunctions or activities of the corresponding full length protein. Thefragment preferably includes at least 20-100 contiguous amino acidresidues of the full length immunoglobulin.

The potential of antibodies as therapeutic modalities is currentlylimited by the production capacity and excessive cost of the currenttechnology. An improved viral or non-viral single expression vector forimmunoglobulin production would permit the expression and delivery oftwo or more coding sequences, i.e., immunoglobulins with bi- ormultiple-specificities from a single vector. The present inventionaddresses these limitations and is applicable to any immunoglobulin(i.e. an antibody) or fragment thereof as further detailed herein,including engineered antibodies such as single chain antibodies,full-length antibodies or antibody fragments.

Antibody Production

In one example of the present invention, the coding sequence for a firstor second chain of a protein or polypeptide is the coding sequence forthe heavy chain or a fragment thereof for any immunoglobulin, e.g., IgG,IgM, IgD, IgE or IgA. Alternatively, the coding sequence for a first orsecond chain of a protein or polypeptide is the coding sequence for thelight chain or a fragment thereof for an IgG, IgM, IgD, IgE or IgA.Genes for whole antibody molecules as well as modified or derived formsthereof, such as fragments, e.g., Fab, single chain Fv(scFv) and F(ab′)₂are include within the scope of the invention. The antibodies andfragments can be animal-derived, human-mouse chimeric, humanized,DeImmunized™ or fully human. The antibodies can be bispecific andinclude but are not limited to diabodies, quadroma, mini-antibodies,ScBs antibodies and knobs-into-holes antibodies.

In practicing the invention, the production of an antibody, or variant(analogue) or fragment thereof using recombinant DNA technology can beachieved by culturing a modified recombinant host cell under cultureconditions appropriate for the growth of that host cell resulting inexpression of the coding sequences. In order to monitor the success ofexpression, antibody levels with respect to the antigen may be monitoredusing standard techniques such as ELISA, RIA, Western blot and the like.The antibodies are recovered from the culture supernatant using standardtechniques known in the art. Purified forms of these antibodies can, ofcourse, be readily prepared by standard purification techniques, e.g.,affinity chromatography via protein A, protein G or protein L columns,or based on binding to the particular antigen, or the particular epitopeof the antigen for which specificity is desired. Antibodies can also bepurified with conventional chromatography, such as an ion exchange orsize exclusion column, in conjunction with other technologies, such asammonia sulfate precipitation and size-limited membrane filtration.Preferred expression systems are designed to include signal peptides sothat the resulting antibodies are secreted into the culture medium orsupernatant, allowing for ease of purification, however, intracellularproduction is also possible.

The production and selection of antigen-specific fully human monoclonalantibodies from mice engineered with human Ig loci, has previously beendescribed (Jakobovits A. et al., Advanced Drug Delivery Reviews Vol. 31,pp: 33-42 (1998); Mendez M, et al., Nature Genetics Vol. 15, pp: 146-156(1997); Jakobovits A. et al., Current Opinion in Biotechnology Vol. 6,No. 5, pp: 561-566 (1995); Green L, et al., Nature Genetics Vol. 7, No.1, pp: 13-21 (1994).

The production and recovery of the antibodies themselves can be achievedin various ways known in the art (Harlow et al., “Antibodies, ALaboratory Manual”, Cold Spring Harbor Lab, 1988).

Vectors for Use in Practicing the Invention

The present invention contemplates the use of any of a variety ofvectors for introduction of constructs comprising the coding sequencefor two or more polypeptides or proteins and a self processing cleavagesequence into cells such that protein expression results. Numerousexamples of expression vectors are known in the art and may be of viralor non-viral origin. Non-viral gene delivery methods which may beemployed in the practice of the invention, include but are not limitedto plasmids, liposomes, nucleic acid/liposome complexes, cationic lipidsand the like.

Viral vectors can efficiently transduce cells and introduce their ownDNA into a host cell. In generating recombinant viral vectors,non-essential genes are replaced with a gene encoding a protein orpolypeptide of interest. Exemplary vectors include but are not limitedto viral and non-viral vectors, such a retroviral vector (includinglentiviral vectors), adenoviral (Ad) vectors including replicationcompetent, replication deficient and gutless forms thereof,adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors,bovine papilloma vectors, Epstein-Barr vectors, herpes vectors, vacciniavectors, Moloney murine leukemia vectors, Harvey murine sarcoma virusvectors, murine mammary tumor virus vectors, Rous sarcoma virus vectorsand nonviral plasmids.

The vector typically comprises an origin of replication and the vectormay or may not in addition comprise a “marker” or “selectable marker”function by which the vector can be identified and selected. While anyselectable marker can be used, selectable markers for use in recombinantvectors are generally known in the art and the choice of the properselectable marker will depend on the host cell. Examples of selectablemarker genes which encode proteins that confer resistance to antibioticsor other toxins include, but are not limited to ampicillin,methotrexate, tetracycline, neomycin (Southern et al., J., J Mol ApplGenet. 1982; 1(4):327-41 (1982)), mycophenolic acid (Mulligan et al.,Science 209:1422-7 (1980)), puromycin, zeomycin, hygromycin (Sugden etal., Mol Cell Biol. 5(2):410-3 (1985)) and G418. As will be understoodby those of skill in the art, expression vectors typically include anorigin of replication, a promoter operably linked to the coding sequenceor sequences to be expressed, as well as ribosome binding sites, RNAsplice sites, a polyadenylation site, and transcriptional terminatorsequences, as appropriate to the coding sequence(s) being expressed.

Reference to a vector or other DNA sequences as “recombinant” merelyacknowledges the operable linkage of DNA sequences that are nottypically operably linked as isolated from or found in nature.Regulatory (expression and/or control) sequences are operatively linkedto a nucleic acid coding sequence when the expression and/or controlsequences regulate the transcription and, as appropriate, translation ofthe nucleic acid sequence. Thus expression and/or control sequences caninclude promoters, enhancers, transcription terminators, a start codon(i.e., ATG) 5′ to the coding sequence, splicing signals for introns andstop codons.

Adenovirus gene therapy vectors are known to exhibit strong transientexpression, excellent titer, and the ability to transduce dividing andnon-dividing cells in vivo (Hitt et al., Adv in Virus Res 55:479-505,2000). The recombinant Ad vectors of the instant invention comprise: (1)a packaging site enabling the vector to be incorporated intoreplication-defective Ad virions; (2) the coding sequence for two ormore proteins or polypeptide of interest; and (3) a sequence encoding aself-processing cleavage site alone or in combination with an additionalproteolytic cleavage site. Other elements necessary or helpful forincorporation into infectious virions, include the 5′ and 3′ Ad ITRs,the E2 genes, portions of the E4 gene and optionally the E3 gene.

Replication-defective Ad virions encapsulating the recombinant Advectors of the instant invention are made by standard techniques knownin the art using Ad packaging cells and packaging technology. Examplesof these methods may be found, for example, in U.S. Pat. No. 5,872,005.The coding sequence for two or more polypeptides or proteins of interestis commonly inserted into adenovirus in the deleted E3 region of thevirus genome. Preferred adenoviral vectors for use in practicing theinvention do not express one or more wild-type Ad gene products, e.g.,E1a, E1b, E2, E3, and E4. Preferred embodiments are virions that aretypically used together with packaging cell lines that complement thefunctions of E1, E2A, E4 and optionally the E3 gene regions. See, e.g.U.S. Pat. Nos. 5,872,005, 5,994,106, 6,133,028 and 6,127,175. Thus, asused herein, “adenovirus” and “adenovirus particle” refer to the virusitself or derivatives thereof and cover all serotypes and subtypes andboth naturally occurring and recombinant forms, except where indicatedotherwise. Such adenoviruses may be wildtype or may be modified invarious ways known in the art or as disclosed herein. Such modificationsinclude modifications to the adenovirus genome that is packaged in theparticle in order to make an infectious virus. Such modificationsinclude deletions known in the art, such as deletions in one or more ofthe E1a, E1 b, E2a, E2b, E3, or E4 coding regions. Exemplary packagingand producer cells are derived from 293, A549 or HeLa cells. Adenovirusvectors are purified and formulated using standard techniques known inthe art.

Adeno-associated virus (AAV) is a helper-dependent human parvovirus thatis able to infect cells latently by chromosomal integration. Because ofits ability to integrate chromosomally and its nonpathogenic nature, AAVhas significant potential as a human gene therapy vector. For use inpracticing the present invention rAAV virions are produced usingstandard methodology, known to those of skill in the art and areconstructed such that they include, as operatively linked components inthe direction of transcription, control sequences includingtranscriptional initiation and termination sequences, and the codingsequence(s) of interest. More specifically, the recombinant AAV vectorsof the instant invention comprise: (1) a packaging site enabling thevector to be incorporated into replication-defective AAV virions; (2)the coding sequence for two or more proteins or polypeptide of interest;(3) a sequence encoding a self-processing cleavage site alone or incombination with an additional proteolytic cleavage site. AAV vectorsfor use in practicing the invention are constructed such that they alsoinclude, as operatively linked components in the direction oftranscription, control sequences including transcriptional initiationand termination sequences. These components are flanked on the 5′ and 3′end by functional AAV ITR sequences. By “functional AAV ITR sequences”is meant that the ITR sequences function as intended for the rescue,replication and packaging of the AAV virion.

Recombinant AAV vectors are also characterized in that they are capableof directing the expression and production of selected recombinantproteins or polypeptides of interest in target cells. Thus, therecombinant vectors comprise at least all of the sequences of AAVessential for encapsidation and the physical structures for infection ofthe recombinant AAV (rAAV) virions. Hence, AAV ITRs for use in thevectors of the invention need not have a wild-type nucleotide sequence(e.g., as described in Kotin, Hum. Gene Ther., 5:793-801, 1994), and maybe altered by the insertion, deletion or substitution of nucleotides orthe AAV ITRs may be derived from any of several AAV serotypes.Generally, an AAV vector is a vector derived from an adeno-associatedvirus serotype, including without limitation, AAV-1, AAV-2, AAV-3,AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, etc. Preferred rAAV expressionvectors have the wild type REP and CAP genes deleted in whole or part,but retain functional flanking ITR sequences.

Typically, an AAV expression vector is introduced into a producer cell,followed by introduction of an AAV helper construct, where the helperconstruct includes AAV coding regions capable of being expressed in theproducer cell and which complement AAV helper functions absent in theAAV expression vector. As used herein, the term “AAV helper functions”refers to AAV coding regions capable of being expressed in the host cellto complement AAV viral functions missing from the rAAV vector.Typically, the AAV helper functions include the AAV rep coding regionand the AAV cap coding region. The helper construct may be designed todown regulate the expression of the large Rep proteins (Rep78 andRep68), typically by mutating the start codon following p5 from ATG toACG, as described in U.S. Pat. No. 6,548,286.

Introduction of an AAV expression vector into a producer cell istypically followed by introduction of helper virus and/or additionalvectors into the producer cell, wherein the helper virus and/oradditional vectors provide accessory functions capable of supportingefficient rAAV virus production.

“Accessory functions” refer to functions that are required by AAV forreplication, but are not provided by the AAV virion itself. Thus, theseaccessory functions and factors must be provided by the host cell, avirus (e.g., adenovirus, herpes simplex virus or vaccinia virus), or byan expression vector that is co-expressed in the same cell. Generally,the E1A and E1B, E2A, E4 and VA coding regions of adenovirus are used tosupply the necessary accessory function required for AAV replication andpackaging (Matsushita et al., Gene Therapy 5:938 [1998]).

The producer cells are then cultured to produce rAAV. These steps arecarried out using standard methodology. Replication-defective AAVvirions encapsulating the recombinant AAV vectors of the instantinvention are made by standard techniques known in the art using AAVpackaging cells and packaging technology. Examples of these methods maybe found, for example, in U.S. Pat. Nos. 5,436,146; 5,753,500,6,040,183, 6,093,570 and 6,548,286. Further compositions and methods forpackaging are described in Wang et al. (US 2002/0168342) and includethose techniques within the knowledge of those of skill in the art. BothAAV vectors and AAV helper constructs can be constructed to contain oneor more optional selectable marker genes. Selectable marker genes whichconfer antibiotic resistance or sensitivity to an appropriate selectivemedium are generally known in the art.

The term “AAV virion” refers to a complete virus particle, such as a“wild-type” (wt) AAV virus particle (comprising a linear,single-stranded AAV nucleic acid genome associated with an AAV capsidprotein coat). In contrast, a “recombinant AAV virion,” and “rAAVvirion” refers to an infectious viral particle containing a heterologousDNA sequence of interest, flanked on both sides by AAV ITRs.

In practicing the invention, host cells for producing rAAV virionsinclude mammalian cells, insect cells, microorganisms and yeast. Hostcells can also be packaging cells in which the AAV rep and cap genes arestably maintained in the host cell or producer cells in which the AAVvector genome is stably maintained and packaged. Exemplary packaging andproducer cells are derived from 293, A549 or HeLa cells. AAV vectors arepurified and formulated using standard techniques known in the art.

Retroviral vectors are also a common tool for gene delivery (Miller,Nature 357: 455-460, 1992). Retroviral vectors and more particularlylentiviral vectors may be used in practicing the present invention.Accordingly, the term “retrovirus” or “retroviral vector”, as usedherein is meant to include “lentivirus” and “lentiviral vectors”respectively. Retroviral vectors have been tested and found to besuitable delivery vehicles for the stable introduction of genes ofinterest into the genome of a broad range of target cells. The abilityof retroviral vectors to deliver unrearranged, single copy transgenesinto cells makes retroviral vectors well suited for transferring genesinto cells. Further, retroviruses enter host cells by the binding ofretroviral envelope glycoproteins to specific cell surface receptors onthe host cells. Consequently, pseudotyped retroviral vectors in whichthe encoded native envelope protein is replaced by a heterologousenvelope protein that has a different cellular specificity than thenative envelope protein (e.g., binds to a different cell-surfacereceptor as compared to the native envelope protein) may also findutility in practicing the present invention. The ability to direct thedelivery of retroviral vectors encoding one or more target proteincoding sequences to specific target cells is desirable in practice ofthe present invention.

The present invention provides retroviral vectors which include e.g.,retroviral transfer vectors comprising one or more transgene sequencesand retroviral packaging vectors comprising one or more packagingelements. In particular, the present invention provides pseudotypedretroviral vectors encoding a heterologous or functionally modifiedenvelope protein for producing pseudotyped retrovirus.

The core sequence of the retroviral vectors of the present invention maybe readily derived from a wide variety of retroviruses, including forexample, B, C, and D type retroviruses as well as spumaviruses andlentiviruses (RNA Tumor Viruses, Second Edition, Cold Spring HarborLaboratory, 1985). An example of a retrovirus suitable for use in thecompositions and methods of the present invention includes, but is notlimited to, a lentivirus. Other retroviruses suitable for use in thecompositions and methods of the present invention include, but are notlimited to, Avian Leukosis Virus, Bovine Leukemia Virus, Murine LeukemiaVirus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma Virus. Preferred MurineLeukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol.19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245),Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma Virus andRauscher (ATCC No. VR-998), and Moloney Murine Leukemia Virus (ATCC No.VR-190). Such retroviruses may be readily obtained from depositories orcollections such as the American Type Culture Collection (“ATCC”;Rockville, Md.), or isolated from known sources using commonly availabletechniques.

Preferably, a retroviral vector sequence of the present invention isderived from a lentivirus. A preferred lentivirus is a humanimmunodeficiency virus, e.g., type 1 or 2 (i.e., HIV-1 or HIV-2, whereinHIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III)and acquired immune deficiency syndrome (AIDS)-related virus (ARV)), oranother virus related to HIV-1 or HIV-2 that has been identified andassociated with AIDS or AIDS-like disease. Other lentiviruses include asheep Visna/maedi virus, a feline immunodeficiency virus (FIV), a bovinelentivirus, simian immunodeficiency virus (SIV), an equine infectiousanemia virus (EIAV), and a caprine arthritis-encephalitis virus (CAEV).

The various genera and strains of retroviruses suitable for use in thecompositions and methods are well known in the art (see, e.g., FieldsVirology, Third Edition, edited by B. N. Fields et al., Lippincott-RavenPublishers (1996), see e.g., Chapter 58, Retroviridae: The Viruses andTheir Replication, Classification, pages 1768-1771, including Table 1.

The present invention provides retroviral packaging systems forgenerating producer cells and producer cell lines that produceretroviruses, and methods of making such packaging systems. Accordingly,the present invention also provides producer cells and cell linesgenerated by introducing a retroviral transfer vector into suchpackaging systems (e.g., by transfection or infection), and methods ofmaking such packaging cells and cell lines.

The packaging systems of the present invention comprise at least twopackaging vectors, a first packaging vector which comprises a firstnucleotide sequence comprising a gag, a pol, or gag and pol genes and asecond packaging vector which comprises a second nucleotide sequencecomprising a heterologous or functionally modified envelope gene. In apreferred embodiment, the retroviral elements are derived from alentivirus, such as HIV. Preferably, the vectors lack a functional tatgene and/or functional accessory genes (vif, vpr, vpu, vpx, nef). In afurther preferred embodiment, the system further comprises a thirdpackaging vector that comprises a nucleotide sequence comprising a revgene. The packaging system can be provided in the form of a packagingcell that contains the first, second, and, optionally, third nucleotidesequences.

The invention is applicable to a variety of systems, and those skilledin the art will appreciate the common elements shared across differinggroups of retroviruses. The description herein uses lentiviral systemsas a representative example. However, all retroviruses share thefeatures of enveloped virions with surface projections and containingone molecule of linear, positive-sense single stranded RNA, a genomeconsisting of a dimer, and the common proteins gag, pol and env.

Lentiviruses share several structural virion proteins in common,including the envelope glycoproteins SU (gp120) and TM (gp41), which areencoded by the env gene; CA (p24), MA (p17) and NC (p7-11), which areencoded by the gag gene; and RT, PR and IN encoded by the pol gene.HIV-1 and HIV-2 contain accessory and other proteins involved inregulation of synthesis and processing virus RNA and other replicativefunctions. The accessory proteins, encoded by the vif, vpr, vpu/vpx, andnef genes, can be omitted (or inactivated) from the recombinant system.In addition, tat and rev can be omitted or inactivated, e.g., bymutation or deletion.

First generation lentiviral vector packaging systems provide separatepackaging constructs for gag/pol and env, and typically employ aheterologous or functionally modified envelope protein for safetyreasons. In second generation lentiviral vector systems, the accessorygenes, vif, vpr, vpu and nef, are deleted or inactivated. Thirdgeneration lentiviral vector systems are preferred for use in practicingthe present invention and include those from which the tat gene has beendeleted or otherwise inactivated (e.g., via mutation).

Compensation for the regulation of transcription normally provided bytat can be provided by the use of a strong constitutive promoter, suchas the human cytomegalovirus immediate early (HCMV-IE)enhancer/promoter. Other promoters/enhancers can be selected based onstrength of constitutive promoter activity, specificity for targettissue (e.g., a liver-specific promoter), or other factors relating todesired control over expression, as is understood in the art. Forexample, in some embodiments, it is desirable to employ an induciblepromoter such as tet to achieve controlled expression. The gene encodingrev is preferably provided on a separate expression construct, such thata typical third generation lentiviral vector system will involve fourplasmids: one each for gagpol, rev, envelope and the transfer vector.Regardless of the generation of packaging system employed, gag and polcan be provided on a single construct or on separate constructs.

Typically, the packaging vectors are included in a packaging cell, andare introduced into the cell via transfection, transduction orinfection. Methods for transfection, transduction or infection are wellknown by those of skill in the art. A retroviral/lentiviral transfervector of the present invention can be introduced into a packaging cellline, via transfection, transduction or infection, to generate aproducer cell or cell line. The packaging vectors of the presentinvention can be introduced into human cells or cell lines by standardmethods including, e.g., calcium phosphate transfection, lipofection orelectroporation. In some embodiments, the packaging vectors areintroduced into the cells together with a dominant selectable marker,such as neo, DHFR, Gln synthetase or ADA, followed by selection in thepresence of the appropriate drug and isolation of clones. A selectablemarker gene can be linked physically to genes encoding by the packagingvector.

Stable cell lines, wherein the packaging functions are configured to beexpressed by a suitable packaging cell, are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al., Proc. Natl. Acad. Sci. (1996)93:11400-11406, which describe packaging cells. Further description ofstable cell line production can be found in Dull et al., 1998, J.Virology 72(11):8463-8471; and in Zufferey et al., 1998, J. Virology72(12):9873-9880.

Zufferey et al., 1997, Nature Biotechnology 15:871-875, teach alentiviral packaging plasmid wherein sequences 3′ of pol including theHIV-1 envelope gene are deleted. The construct contains tat and revsequences and the 3′ LTR is replaced with poly A sequences. The 5′ LTRand psi sequences are replaced by another promoter, such as one which isinducible. For example, a CMV promoter or derivative thereof can beused.

Preferred packaging vectors may contain additional changes to thepackaging functions to enhance lentiviral protein expression and toenhance safety. For example, all of the HIV sequences upstream of gagcan be removed. Also, sequences downstream of the envelope can beremoved. Moreover, steps can be taken to modify the vector to enhancethe splicing and translation of the RNA.

Optionally, a conditional packaging system is used, such as thatdescribed by Dull et al., J. Virology 72(11):8463-8471, 1998. Alsopreferred is the use of a self-inactivating vector (SIN), which improvesthe biosafety of the vector by deletion of the HIV-1 long terminalrepeat (LTR) as described, for example, by Zufferey et al., 1998, J.Virology 72(12):9873-9880. Inducible vectors can also be used, such asthrough a tet-inducible LTR.

Any vector for use in practicing the invention will include heterologouscontrol sequences, such as a constitutive promoter, e.g., thecytomegalovirus (CMV) immediate early promoter, the RSV LTR, the MoMLVLTR, and the PGK promoter; tissue or cell type specific promotersincluding mTTR, TK, HBV, hAAT, regulatable or inducible promoters,enhancers, etc. Preferred promoters include the LSP promoter (III etal., Blood Coagul. Fibrinolysis 8S2:23-30, 1997), the EF1-alpha promoter(Kim et al., Gene 91(2):217-23, 1990) and Guo et al., Gene Ther.3(9):802-10, 1996). Most preferred promoters include the elongationfactor 1-alpha (EF1a) promoter, a phosphoglycerate kinase-1 (PGK)promoter, a cytomegalovirus immediate early gene (CMV) promoter,chimeric liver-specific promoters (LSPs), a cytomegalovirusenhancer/chicken beta-actin (CAG) promoter, a tetracycline responsivepromoter (TRE), a transthyretin promoter (TTR), an simian virus 40(SV40) promoter and a CK6 promoter. The sequences of these and numerousadditional promoters are known in the art. The relevant sequences may bereadily obtained from public databases and incorporated into vectors foruse in practicing the present invention.

The present invention also contemplates the inclusion of a generegulation system for the controlled expression of the coding sequencefor two or more polypeptides or proteins of interest. Gene regulationsystems are useful in the modulated expression of a particular gene orgenes. In one exemplary approach, a gene regulation system or switchincludes a chimeric transcription factor that has a ligand bindingdomain, a transcriptional activation domain and a DNA binding domain.The domains may be obtained from virtually any source and may becombined in any of a number of ways to obtain a novel protein. Aregulatable gene system also includes a DNA response element whichinteracts with the chimeric transcription factor. This element islocated adjacent to the gene to be regulated.

Exemplary gene regulation systems that may be employed in practicing thepresent invention include, the Drosophila ecdysone system (Yao et al.,Proc. Nat. Acad. Sci., 93:3346 (1996)), the Bombyx ecdysone system (Suhret al., Proc. Nat. Acad. Sci., 95:7999 (1998)), the Valentis GeneSwitch®synthetic progesterone receptor system which employs RU-486 as theinducer (Osterwalder et al., Proc Natl Acad Sci 98(22):12596-601(2001)); the Tet™ & RevTet™ Systems (BD Biosciences Clontech), whichemploys small molecules, such as tetracycline (Tc) or analogues, e.g.doxycycline, to regulate (turn on or off) transcription of the target(Knott et al., Biotechniques 32(4):796, 798, 800 (2002)); ARIADRegulation Technology which is based on the use of a small molecule tobring together two intracellular molecules, each of which is linked toeither a transcriptional activator or a DNA binding protein. When thesecomponents come together, transcription of the gene of interest isactivated. Ariad has two major systems: a system based onhomodimerization and a system based on heterodimerization (Rivera etal., Nature Med, 2(9):1028-1032 (1996); Ye et al., Science 283: 88-91(2000)), either of which may be incorporated into the vectors of thepresent invention.

Preferred gene regulation systems for use in practicing the presentinvention are the ARIAD Regulation Technology and the Tet™ & RevTet™Systems.

Delivery of Nucleic Acid Constructs Including Protein or PolypeptideCoding Sequences to Cells

The vector constructs of the invention comprising nucleic acid sequencesencoding heterologous proteins or polypeptides, and a self-processingcleavage site alone or in combination with a sequence encoding anadditional proteolytic cleavage site may be introduced into cells invitro, ex vivo or in vivo for expression of heterologous codingsequences by cells, e.g., somatic cells in vivo, or for the productionof recombinant polypeptides by vector-transduced cells, in vitro or invivo.

The vector constructs of the invention may be introduced into cells invitro or ex vivo using standard methodology known in the art. Suchtechniques include transfection using calcium phosphate, microinjectioninto cultured cells (Capecchi, Cell 22:479-488 (1980)), electroporation(Shigekawa et al., BioTechn., 6:742-751 (1988)), liposome-mediated genetransfer (Mannino et al., BioTechn., 6:682-690 (1988)), lipid-mediatedtransduction (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417(1987)), and nucleic acid delivery using high-velocity microprojectiles(Klein et al., Nature 327:70-73 (1987)).

For in vitro or ex vivo expression, any cell effective to express afunctional protein may be employed. Numerous examples of cells and celllines used for protein expression are known in the art. For example,prokaryotic cells and insect cells may be used for expression. Inaddition, eukaryotic microorganisms, such as yeast may be used. Theexpression of recombinant proteins in prokaryotic, insect and yeastsystems are generally known in the art and may be adapted for protein orpolypeptide expression using the compositions and methods of the presentinvention.

Exemplary host cells useful for expression further include mammaliancells, such as fibroblast cells, cells from non-human mammals such asovine, porcine, murine and bovine cells, insect cells and the like.Specific examples of mammalian cells include COS cells, VERO cells, HeLacells, Chinese hamster ovary (CHO) cells, 293 cell, NSO cells, 3T3fibroblast cells, W138 cells, BHK cells, HEPG2 cells, DUX cells and MDCKcells.

Host cells are cultured in conventional nutrient media, modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. Mammalian hostcells may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium (MEM), Sigma),RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma)are typically suitable for culturing host cells. A given medium isgenerally supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics, trace elements, and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theappropriate culture conditions for a particular cell line, such astemperature, pH and the like, are generally known in the art, withsuggested culture conditions for culture of numerous cell lines forexample in the ATCC Catalogue available on line at“http://www.atcc.org/SearchCatalogs/AllCollections.cfm”

The vectors of the invention may be administered in vivo via variousroutes (e.g., intradermally, intravenously, intratumorally, into thebrain, intraportally, intraperitoneally, intramuscularly, into thebladder etc.), to deliver multiple genes connected via a self processingcleavage sequence to express two or more proteins or polypeptides inanimal models or human subjects. Dependent upon the route ofadministration, the therapeutic proteins elicit their effect locally(e.g., in brain or bladder) or systemically (other routes ofadministration). The use of tissue specific promoters 5′ to the openreading frame(s) for a protein or polypeptide in the vectors of theinvention may be used to effect tissue specific expression of the two ormore proteins or polypeptides encoded by the vector.

Various methods that introduce a recombinant vector carrying a transgeneinto target cells in vitro, ex vivo or in vivo have been previouslydescribed and are well known in the art. The present invention providesfor therapeutic methods, vaccines, and cancer therapies by transducingtarget cells with recombinant vectors of the invention.

For example, in vivo delivery of the recombinant vectors of theinvention may be targeted to a wide variety of organ types including,but not limited to brain, liver, blood vessels, muscle, heart, lung andskin.

In the case of ex vivo gene transfer, the target cells are removed fromthe host and genetically modified in the laboratory using recombinantvectors of the present invention and methods well known in the art.

The recombinant vectors of the invention can be administered usingconventional modes of administration including but not limited to themodes described above. The recombinant vectors of the invention may beprovided in any of a variety of formulations such as liquid solutionsand suspensions, microvesicles, liposomes and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application. A from appropriate to the route ofdelivery may be readily determined using knowledge generally availableto those of skill in the relevant art.

The many advantages to be realized in using the inventive recombinantvector constructs of the invention in recombinant protein andpolypeptide production in vivo include administration of a single vectorfor long-term and sustained expression of two or more recombinantprotein or polypeptide ORFs in patients; in vivo expression of two ormore recombinant protein or polypeptide ORFs having biological activity;and the natural posttranslational modifications of the recombinantprotein or polypeptide generated in human cells.

One preferred aspect is use of the recombinant vector constructs of thepresent invention for the in vitro production of recombinant proteinsand polypeptides. Methods for recombinant protein production are wellknown in the art and self processing cleavage site-containing vectorconstructs of the present invention may be utilized for expression ofrecombinant proteins and polypeptides using such standard methodology.

In one exemplary aspect of the invention, vector introduction oradministration to a cell (transfection) is followed by one or more ofthe following steps:

(1) culturing the transfected cell under conditions to selecting for acell expressing the recombinant protein or polypeptide;

(2) evaluating expression of the recombinant protein or polypeptide; and

(3) collecting the recombinant protein or polypeptide.

The objects of the invention have been achieved by a series ofexperiments, some of which are described by way of the followingnon-limiting examples.

EXAMPLES Example 1 Expression of Two Secreted Proteins Using the FMDV 2Aand IRES Sequences

In one exemplary application of the method described herein, the codingsequences for VEGF-TRAP and platelet factor 4 (PF4) were expressed froma single promoter using either a 2A sequence or an IRES. At present, theIRES represents the state of the art for expressing two proteins from asingle promoter. The purpose of this experiment was to compare theprotein expression from a vector containing a 2A or 2A-like sequence tothat from a vector containing an IRES. A schematic depiction of theplasmids used in this experiment is provided in FIGS. 1A-D. Vectorcomponents include the following:

CAG promoter The cytomegalovirus enhancer/chicken beta-actin/Rabbitβ-globin promoter (CAG promoter; Niwa H. et al. 1991. Gene 108(2):193-9) 2A SEQ ID NO: 14 Furin SEQ ID NO: 15 WPRE (Woodchuck HepatitisSEQ ID NO: 16 Virus Post-Transcriptional Regulatory Element) bGHpolyA(Bovine Growth SEQ ID NO: 17 Hormone Polyadenylation Signal Sequence)EMCV IRES A 492 base pair IRES obtainable from encephelomycarditis virus(EMCV) SEQ ID NO: 18 sPF4 pBLAST-hPF4 (Invivogen) and Tanaka, T. et al.Nat. Med. 3(4): 437-42, 1997. The hPF4 sequence was modified to containa DLR mutation (Hagedorn et al. Cancer Res. 62: 6884-6890, 2002). Themutation was generated using a Quick-Change Site-Directed MutagenesisKit (Stratagene) and the following oligonucleotide primers: sPF4(DLR)FOR: SEQ ID NO: 19 sPF4(DLR) REV: SEQ ID NO: 20 VEGF-TRAP R1 D2 FOR: SEQID NO: 21 R1 D2 REV: SEQ ID NO: 22 R2 D3 FOR: SEQ ID NO: 23 R2 D3 REV:SEQ ID NO: 24 FC FOR: SEQ ID NO: 25 FC REV: SEQ ID NO: 26 The signalsequence for VEGFR1 was generated using the oligonucleotides shown belowand fused to the aforementioned chimeric fragment using standardmolecular biology techniques. R1 SS FOR: SEQ ID NO: 27 R1 SS REV: SEQ IDNO: 28 sPF4:F2A: The sPF4 (DLR) and VEGF-TRAP coding sequences wereinitially cloned VEGF TRAP downstream of the CAG expression cassette assingle proteins. These and plasmids were used as the basis forconnecting the two proteins using the VEGF TRAP: F2A:sPF4 F2A sequence,which was performed by PCR SOEing using the following oligonucleotideprimers: PF4-F2A FOR: SEQ ID NO: 29 PF4-F2A REV: SEQ ID NO: 30 F2A-VTFOR: SEQ ID NO: 31 F2A-VT REV: SEQ ID NO: 32 PF4 FOR: SEQ ID NO: 33 VTREV: SEQ ID NO: 34 VEGF TRAP:EMCV IRES:sPF4 The ECMV IRES was PCRamplified with the primers listed below and and cloned in between thesPF4 (DLR) and VEGF-TRAP nucleotide sequences sPF4: EMCV IRES:VEGF TRAPusing standard molecular biology procedures. PF4-I-VT REV: SEQ ID NO: 35VT-I-PF4 REV: SEQ ID NO: 36 IRES FOR: SEQ ID NO: 37In these examples the 2A or IRES sequences were placed between theVEGF-TRAP and sPF4 coding sequence and the complete cassette was drivenby the CAG promoter. Both orientations of the two genes relative to theF2A and IRES sequences were cloned and evaluated (FIGS. 1A-D). Theseplasmids were initially tested by transient transfection into 293Tcells, which was performed in a 6-well dish using a FUGENE 6 kit(Roche). The transfections were done in triplicate using 2×10⁵ cells andlog of DNA per well, and 200 ng of a CAG-GFP expressing plasmid wasadded to each sample as a transfection control. Cell culturesupernatants were harvested approximately 40 hours later and assayed forPF4 expression using an Asserachrom PF4 ELISA assay (Diagnostica Stago).The VEGF-TRAP protein contains domain 2 of VEGFR1 and domain 3 of VEGFR2connected to the human IgG Fc domain, and is detected with a human IgGELISA kit (Bethyl Laboratories) using recombinant VEGFR1-Fc (R & DSystems) to generate a standard curve. The cells were harvested andsubjected to FACs analysis for GFP expression. The results of theseassays are shown in FIGS. 2A-C. In the IRES containing vectors, the geneupstream of the IRES is expressed at high levels whereas the genedownstream of the IRES is very poorly expressed. This is in strikingcontrast to the F2A containing vectors, which expressed both proteins atequal levels. Expression levels of the two proteins are almost identicalwhen placed either upstream or downstream F2A site, indicating that theF2A sequence appears to function independent of orientation/position.These data show that the F2A provides a significant improvement over thestate of the art in expressing two genes from a single promoter.

Example 2 Expression of Human Factor VIII from a 2A Construct

We previously demonstrated that the 2A sequence can be used toefficiently express the heavy and light chains of human and ratmonoclonal antibodies. (See, e.g., U.S. Ser. No. 60/540,554.) In thisexample a 2A sequence was evaluated as for its ability to express theheavy and light chains human of Factor VIII using a single promoter. Atypical Factor VIII expression construct includes a promoter, a FactorVIII heavy chain coding sequence, a furin cleavage site (RAKR), a 2Asequence, a Factor VIII light chain coding sequence, and a polyAsequence (FIG. 3). FIG. 4 shows four exemplary methods of linking theheavy and light chains of human Factor VIII to a self-processingcleavage site (e.g., 2A) with and without an additional proteolyticcleavage site (e.g., RAKR) are under evaluation. These constructs weredesigned to express a B domain deleted form of Factor VIII based on the‘SQ’ B-domain deletion (Lind et al., Eur. J. Biochem. 232:19-27, 1995),which fuses Ser 743 to Glu 1638, and is identical to the B-domaindeletion in Refacto, a licensed recombinant Factor VIII product(Eriksson et al., Semin. Hematol. 38:24-31, 2001). In the native FactorVIII protein, QN is repeated at amino acids 744 and 1638. For thesestudies, QN was left at the 5′ end of the B-domain deletion. Previousdata where Factor VIII heavy and light chains were separately expressedindicated that the heavy chain was more stable with the addition ofthese extra two amino acids (Yonemura et al., Protein Eng. 6: 669-674(1993)). In each of the exemplary constructs shown in FIG. 4, a humanIgG signal peptide (SS) is cloned upstream of the light chain. It haspreviously been shown that when joining two proteins with a selfprocessing sequence such as 2A, the C-terminal protein requires a signalsequence for efficient secretion (de Felipe et al., J. Biol. Chem.278:11441-11448, 2003). In construct D1, both the endogenous Factor VIIIcleavage sites, as well as an additional furin (RAKR) and 2A cleavagesite are present. The D2 and E1 constructs have the endogenous furin andthrombin cleavage sites removed, respectively. The E2 construct containsboth endogenous cleavage sites and a 2A sequence, but is missing theadditional furin site.

The processing of the D1 construct is diagrammed in FIG. 5. The 2A siteis initially cleaved during protein translation. The signal sequence issubsequently cleaved and the furin cleavage and removal of the 2Asequence can then takes place due to the presence of the RAKR sequence.The final thrombin cleavage and Factor VIII activation typically occurin the plasma. A cell line expressing this construct will produce FactorVIII recombinant protein that does not contain any additional aminoacids.

When these constructs are transiently transfected into cell lines, e.g.,CHO, BHK, and 293, cell culture supernatants and lysates are examinedfor Factor VIII activity using the Coamatic assay, Factor VIII proteinexpression by ELISA, and Factor VIII cleavage and secretion by Westernblot analysis. These analyses are generally typically used by those ofskill in the art to evaluate the relative efficiency of expression,cleavage, and secretion of Factor VIII protein.

Example 3 Expression of a Rat IgG from an AAV H2AL Plasmid Transfectedinto 293 T Cells

An AAV plasmid (pAAV H2AL) encoding the heavy and light chain of amonoclonal IgG antibody against murine FLK-1 and linked by insertion ofthe FMDV 2A sequence (FIG. 6), was transiently transfected into 293Tcells. Cells were grown in Iscove's Modified Dulbecco's Medium (IMDM)supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1%penicillin-streptomycin solution (Invitrogen). Transfection was carriedout using a FuGENE 6 transfection kit (Roche). pAAV H2AL plasmid DNA wasmixed with the transfection reagent according to the manufacturer'sinstruction and the DNA-lipid mixture was added to the cell culturemedium. The transfected cells were incubated for 48 or 72 hours and thesupernatants analyzed for antibody expression. The mAb concentration wasdetermined using a rat IgG ELISA assay (Bethyl Laboratories), in whichmAb IgG protein was captured by an immobilized anti-rat IgG antibody onELISA plates and detected by an anti-rat IgG Fc antibody conjugated withHRP. The ELISA plates were developed and mAb concentrations werecalculated based on OD reading of the samples as compared to a standardcurve with known rat IgG concentrations. ELISA assay results revealedthat the recombinant rat IgG antibody was expressed at high levels inthe supernatant of 293T cells transfected with the AAV plasmidcontaining a 2A sequence (FIG. 7).

The biological activity of the antibody was evaluated for neutralizingactivity in a VEGF-FLK-1 binding assay. In this assay, recombinant VEGF(vascular endothelial cell growth factor, from R & D Systems) was coatedon ELISA plates (Nunc), then blocked with 5% milk. The rat anti-FLK-1antibody was pre-incubated at various concentrations with recombinantFLK-1-Fc (R & D Systems). The antibody/FLK-1 mixture was transferred toELISA wells and the plates were incubated to allow VEGF-FLK-1 binding.After rinsing with balance solution, a goat anti-FLK-1 antibodyconjugated with biotin was used to detect bound FLK-1, which wasvisualized by streptavidin-HRP (PharMingen) after color development withthe HRP substrate.

By using the VEGF/FLK-1 (ligand-receptor) binding assay, it wasdemonstrated that the antibody expressed from 293T cells followingtransient transfection exhibits full biological activity, similar tothat of the native antibody expressed by parent hybridoma cells (FIG.8).

The antibody expressed from the plasmid utilizing the self processing 2Asequence was further characterized using Western blot analysis. Proteinin the supernatant of transiently transfected 293T cells (transfectedwith AAV H2AL plasmid) or from that of hybridoma cells was separated bypolyacrylamide gel electrophoresis under reducing or non-reducingconditions. For the reducing gel, protein samples were mixed with 2×LDSsample buffer (Invitrogen), boiled, loaded on pre-cast 12% Tris-Glycinegel (Invitrogen), and run with Tris-Glycine SDS running buffer. For thenon-reducing gel, protein samples were mixed with 2× native TrisGlysample buffer (Invitrogen), loaded on pre-cast 12% Tris-Glycine gel(Invitrogen), and run with Tris-Glycine native running buffer(Invitrogen). After electrophoresis, the proteins were transferred tonitrocellulose membranes in Tris-Glycine transfer buffer with 20%methanol. The membranes were blocked with blocking solution and stainedwith HRP-conjugated anti-rat IgG. The membrane blots were treated usingreagents provided in a SuperSignal West Chemiliminescent substrate kit(Pierce) and protein bands were visualized in Biome film (Kodak).

Western blot analysis revealed that the antibodies from both theparental hybridoma cell line and the transfected 293T cells appear as anapproximately 160 kD band on a non-reducing gel (FIG. 9A). Thisindicates that the heavy and light chains generated via the 2A cleavagesite dimerize properly with a heavy and light chain ratio of 1:1, giventhat no additional bands, such as an approximately 133 kD band whichwould indicate a heavy to light chain ratio of 2:1, were detected. On areducing gel, the antibodies from both hybridoma and transfected 293Tcells appeared as an approximately 55 kD band (heavy chain) and a 23 kDband (light chain). No uncleaved 78 kD precursor polyprotein wasdetected, indicating efficient cleavage by the 2A peptide (FIG. 9B).Antibody expressed from the H2AL construct appeared to have a slightlylarger molecular weight, which may be due to the additional amino acidresidues contributed by the 2A sequence.

These results demonstrate that the 2A sequence provided a “cleavage”site facilitating the generation of both chains of the IgG moleculeduring the translation process in 293T cells. In other words, thechimeric H2AL polyprotein underwent autolytic cleavage to yield a fulllength, intact Ig molecule containing two heavy chains and two lightchains following dimerization.

Example 4 Expression of a Human Immunoglobulin from an AAV H2ALConstruct

In another example used to illustrate the invention, an AAV constructcomprising a self processing cleavage site was used to express the heavyand light chain of a human monoclonal antibody directed to KDR. An AAVvector comprising a sequence encoding a novel human anti-VEGFR2 (KDR)mAb heavy chain, a sequence encoding a self-processing 2A cleavage site,and a sequence encoding a human anti-VEGFR2 (KDR) mAb light chain wasconstructed using the same strategy described in Example 3. The AAVvector also contains an EF1-alpha or CAG promoter, a WPRE, and poly Asequence. 293T cells were transfected with the AAV plasmid using aFuGENE 6 kit based on the manufacture's instructions and cellsupernatants were harvested 48 or 72 hours post-transfection. Theconcentration of the mAb in 293T cell supernatants was determined usinga sandwich ELISA assay for human IgG (Bethyl Laboratories). In thisassay, human IgG was captured by an immobilized anti-human IgG antibodyon ELISA plates and detected by an anti-human IgG Fc antibody conjugatedwith HRP. Color was developed after adding substrate solution to thewells and mAb concentrations were calculated based on OD reading of thesamples with the human IgG of known concentrations as a standard curve.

The results demonstrate that transfection of an AAV plasmid encoding theheavy and light chains of a human antibody linked by a sequence encodinga self-processing 2A cleavage site into 293T cells resulted in highlevel expression of a full length antibody in cell culture supernatants(FIG. 10). These results show that heavy and light chains of a humanantibody can be generated from a single open reading frame using avector comprising a sequence encoding a self-processing 2A cleavage sitewhich results in autocleavage. Furthermore, the heavy and light chainsare folded and secreted properly.

Example 5 In Vivo Expression of a Full-Length Rat Anti-Flk-1 MonoclonalAntibody by a 2A Self-Processing Sequence Containing AAV Vector

In another example of the invention, two polypeptides, specifically IgGheavy and light chains of a rat anti-FLK-1 monoclonal antibody, wereexpressed in vivo from a single promoter using a rAAV vector. Deliveryof monoclonal antibodies by gene therapy has a number of advantagesrelative to conventional methods currently used in the clinic. rAAV is apreferred gene therapy viral system due to its safety profile andsustained gene expression. In this invention, a rAAV-6 vector wasconstructed to contain an AAV ITR, a CAG promoter, and polyA sequences.The vector includes a single open reading frame comprising in the 5′ to3′ direction, the coding sequence for a rat IgG heavy chain, the codingsequence for a self processing 2A sequence, and the coding sequence foran antibody light chain, engineered and cloned into the vectoroperatively linked to the CAG promoter. The total size of the rAAVconstruct is within the size limit of rAAV and viral particles areeffectively packaged as demonstrated by viral production in 293 cells.

Replication-deficient rAAV virus was generated in 293 cells using AAVplasmid transfection in the presence of adenovirus. rAAV viruses werepurified by CsCI2 gradient centrifugation and the physical titersdetermined by dot blot. Purified rAAV was used to infect 293 cells orU87 glioma cells and monoclonal antibody concentrations in supernatantswere measured by a rat IgG ELISA as described in Example 3.

For antibody expression in vivo, 2×10¹¹ viral particles wereadministered by intramuscular (IM) injection into mice. Mice were bledat various time points. The monoclonal antibody level in serum wasquantified using the rat IgG ELISA assay described in Example 3. Theresults showed that high levels of monoclonal antibodies were detectedfollowing IM injection of rAAV-6 that encodes full length rat anti-FLK-1antibody (FIG. 11). Expression reached a maximum level of 4.5 ug/ml anda stable expression level of about 2.5 □g/ml was detected. The highestserum antibody level detected in any individual mouse was above 9 ug/ml(day 21).

These results demonstrate that full length mAb heavy and light chainproteins were successfully expressed at high levels in mice by use ofvectors comprising a 2A self-processing sequence, demonstrating that 2Aself-processing sequence mediated separation of antibody heavy and lightchains takes place in vivo consistent with in vitro expression.Accordingly, the present invention provides a means to delivertherapeutic antibodies to patients in vivo in order to achieve a longterm therapeutic effect. The expression cassette used in this study caneasily be adapted to other vector systems, such as lentivirus,adenovirus, etc, using routine technology routinely employed by those ofskill in the art.

Example 6 Removal of 2A Cleavage Site Residues from Antibodies Expressedvia an AAV HF2AL Vector

Antibody heavy chains expressed using the H2AL constructs describedabove carry amino acid residues derived from the self processingcleavage sequence such as a 2A or 2A-like sequence at their C-terminus,which remain following self cleavage. To further optimize the expressionsystem of the invention, a vector/plasmid was constructed which includesa protease cleavage site between the first polypeptide, i.e. theantibody heavy chain in this particular construct, and the selfprocessing 2A sequence. The cleavage site used in the construct was RAKR(SEQ ID NO: 11), which belongs to the category of furin cleavageconsensus sequences RXK(R)R (SEQ ID NO:10). Expected cleavage occursbetween A and K in this cleavage site by furin or other proteases. Theconstruct consists The construct comprises in the 5′ to 3′ direction: aCAG promoter, an antibody heavy chain coding sequence, a furin cleavagesite coding sequence, a 2A cleavage site coding sequence, an antibodylight chain coding sequence, and a polyA sequence (CAG HF2AL) (FIG. 12).

To express the antibody from the CAG HF2AL construct, plasmid DNA waspurified using a Qiagen plasmid DNA purification kit and used totransfect 293T cells in 6 well tissue culture plates using the FuGENE 6kit (Roeche). The next day, cells were fed with serum-free medium andthe conditioned media were harvested after 48 hours. In one controlexperiment, 293T cells were transfected with H2AL plasmid, whichcontains the same antibody and 2A sequence but lacks the furin cleavagesite between the heavy chain and the 2A sequence. In the second controlexperiment, 293T cells were transfected with HFL plasmid, which containsthe antibody heavy chain, the furin cleavage site, and the antibodylight chain, but lacks the 2A sequence. Antibody concentrations inconditioned media were determined by ELISA. As shown in FIG. 13, theHF2AL construct gave higher antibody expression levels in supernatantsfrom transfected cells than the H2AL construct. On the other hand, onlyvery limited amount of antibody was detected in 293T cell supernatanttransfected with the HFL (heavy chain-furin-light chain) construct.

The efficiency of removal of the additional 2A amino acid residues fromthe heavy chain of the antibody using a furin cleavage site wasevaluated by separating antibodies in supernatants of HF2AL and H2ALtransfected cells on a 12% Tris-Glycine SDS-PAGE gel under reducingconditions. The separated proteins were transferred onto anitrocellulose membrane and the protein band for the antibody heavychain was detected by a rabbit anti-rat antibody. This Western blotanalysis showed that the antibody heavy chains expressed from the HF2ALplasmid in 293T cells migrated as a single band at a molecular weightthat was smaller than the heavy chains expressed from the H2AL constructbut similar to the antibody heavy chains expressed by parental hybridomacells. This result suggests that the furin cleavage site within theHF2AL construct provides an efficient means to remove residual 2Aderived amino acids.

Example 7 Expression of Antibodies in Furin −/− Cells FollowingTransfection with AAV Plasmids Containing a 2A Site and Furin CleavageSite

Furin is a ubiquitous subtilisin-related serine protease that isexpressed in almost all cell types.

Two cell lines, LoVo and CHO mutant RPE.40, have been found to have nofunctional furin due to mutations. Given that the furin cleavage siteRAKR used in the CAG HF2AL construct (Example 6) can be cleaved by furinas well as many other members of proteases in the same family, anexperiment was conducted to identify the actual enzyme responsible forthe cleavage of RAKR in the antibody expressed from the CAG HF2ALconstruct. Plasmids with or without a furin cleavage site (HF2AL orH2AL) were used to transfect LoVo cells. LoVo is a human colon carcinomacell line with no functional furin due to one nucleotide deletion in theregion covering the homo B domain essential for the endoproteolyticactivity to RXK(R)R (Takahashi et al., Biochem Biophys Res Commun. 195:1019-26. (1993)).

Following HF2AL and H2AL plasmid transfection into LoVo cells using theFuGENE 6 kit, cell culture supernatants were harvested from tissueculture dishes. Proteins were separated in a 12% Tris-Glycine SDS-PAGEgel under reducing conditions and analyzed in Western blot analysis, asdescribed in Example 3. Results showed that the antibody heavy chainsexpressed from the HF2AL plasmid migrated at a molecular weight similarto the heavy chains expressed from the H2AL construct but higher thanthe antibody heavy chain expressed by parental hybridoma cells (FIG.14). These results demonstrate that in LoVo cells which lack furinactivity, additional amino acids derived from the 2A cleavage siteremain at the C-terminus of the antibody heavy chain, confirming thatthe protease furin is the actual enzyme responsible for removal of 2Aresidues from the antibody when expressed in furin containing cells,such as 293T cells.

To further confirm the removal of residual amino acids from the 2Apeptide sequence at the C terminus of the heavy chain expressed from theHF2A vector, the C-terminal fragment of the antibody heavy chain wasanalyzed by mass spectrum analysis. An expression vector was constructedthat contains the rat antibody heavy chain, a furin cleavage siteadjacent to the 2A cleavage site (RAKR), the antibody light chain, and 6his amino acids (HF2AL 6H), called “His-Tag”. The plasmid was injectedinto mice via hydrodynamic gene transfer. The his-tagged monoclonalantibody was purified from mouse serum under native conditions using aNickel column (Qiagen). The antibody heavy and light chains wereseparated on a 10% SDS-PAGE gel stained with Coomassie blue. Theantibody heavy chain band was isolated from the SDS-PAGE gel andsubjected to mass spectrum analysis after trypsin digestion. Massspectrum data confirmed the removal of all but two amino acids derivedfrom the 2A/furin sequences at the C terminus of the antibody heavychain. Furthermore, by using combination of mass spectrum and PSD(MS/MS) sequencing analyses, it could be shown that the antibody heavychain expressed from the HF2AL construct has the C-terminal sequence“SLSHSPGKRA”, which includes native rat IgG heavy chain C-terminal aminoacids plus two additional amino acids (RA) derived from the furincleavage site.

In summary, the results provided herein demonstrate that residual aminoacids derived from a self processing cleavage sequence, such as a 2A or2A-like sequence can be efficiently removed during protein expressionand secretion by introducing an additional proteolytic cleavage site(i.e., a furin cleavage site) adjacent to the 2A cleavage site. Removalof 2A sequence derived amino acids results in generation of a productlacking foreign amino acid residues which could otherwise elicit immuneresponses when administered in vivo. Furthermore, these data suggestthat the addition of a furin cleavage site in 2A containing constructsresults in an overall increase in antibody expression level.

1-27. (canceled)
 28. An oligonucleotide construct comprising, in the 5′to 3′ direction, a promoter operably linked to all of (1) a codingsequence for a first protein, polypeptide or fragment thereof, whereinsaid first protein, polypeptide or fragment thereof is not the lightchain of an immunoglobulin, (2) a coding sequence for a furin cleavagesite, (3) a coding sequence for a 2A self-processing cleavage site and(4) a coding sequence for a second protein, polypeptide or fragmentthereof, wherein said second protein, polypeptide or fragment thereof isnot the heavy chain of an immunoglobulin, and wherein the codingsequence for the furin cleavage site is immediately 5′ to the codingsequence for the 2A self-processing cleavage site.
 29. Theoligonucleotide construct of claim 28 wherein the coding sequence forsaid 2A self-processing cleavage site encodes an oligopeptide selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ IDNO:9.
 30. The oligonucleotide construct of claim 28 wherein the codingsequence for the 2A self processing cleavage site is the oligonucleotidesequence presented as SEQ ID NO:14.
 31. The oligonucleotide construct ofclaim 28 wherein the coding sequence for the 2A self processing cleavagesite encodes the oligopeptide presented as SEQ ID NO:6.
 32. Theoligonucleotide construct of claim 28 wherein said coding sequence forthe furin cleavage site encodes an oligopeptide having the consensussequence presented as SEQ ID NO:10.
 33. The oligonucleotide construct ofclaim 28 wherein said first protein, polypeptide or fragment thereof isan antibody heavy chain.
 34. The oligonucleotide construct of claim 28wherein said second protein, polypeptide or fragment thereof is anantibody light chain.
 35. The oligonucleotide construct of claim 28wherein the promoter is selected from the group consisting of anelongation factor 1-alpha promoter (EF1a) promoter, a phosphoglyceratekinase-1 promoter (PGK) promoter, a cytomegalovirus immediate early genepromoter (CMV), a chimeric liver-specific promoter (LSP) acytomegalovirus enhancer/chicken beta-actin promoter (CAG), atetracycline responsive promoter (TRE), a transthyretin promoter (TTR),a simian virus 40 promoter (SV40) and a CK6 promoter.